Rna products and uses thereof

ABSTRACT

Small RNAs, inhibitors thereof, inhibitors of enzymes producing small RNAs, and the use of these to modulate the response of a cell to a DNA damaging event. A method of detecting the presence of, or of quantifying DNA damage.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSequenceListing.txt, created Feb. 6, 2019, which is 11.6 kb bytes insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to small RNAs (DDRNAs), inhibitorsthereof, inhibitors of enzymes producing thereof, and their use tomodulate the response of a cell to a DNA damaging event. The inventionconcerns also a method to detect the presence or quantify DNA damage.

BACKGROUND ART

The DNA damage response (DDR) is a coordinate set of events thatpromptly follows the generation of a lesion in the DNA double helix.Detection of DNA discontinuities by specialized factors initiates asignaling cascade that, stemming from the site of DNA damage, amplifiesthe signal and reaches the whole nuclear space and the entire cell¹. DDRsignaling cascade initiation establishes a local self-feeding loop thatleads to focal accumulation of upstream DDR factors in the form ofcytologically detectable DDR foci at damaged sites. Specifically,detection of a DNA double-strand break (DSB) triggers the activity ofthe protein kinase ATM that, among other factors, phosphorylates thehistone variant H2AX (γH2AX) at the DNA damage site. This modificationrecruits DDR-mediators like MDC1 and 53BP1 that boost ATM activity. DDRactivation can be triggered by exogenous DNA damaging agents such asionizing radiations and chemotherapeutic agents (i.e. including but notlimited to bleomycin) and by endogenous physiological events such asmeiotic recombination, V(D)J recombination at the immunoglobulins and Tcell receptor loci, telomere shortening and reactive oxygen species, aswell as pathological events such as oncogene activation, viralintegration in the genome, viral replication and bacterialinfection^(1,82). Telomeres dysfunction and oncogene activation cangenerate a sustained DDR leading to a permanent cell-cycle arrest knownas cellular senescence². Recently also bacteria have been shown togenerate persistent DNA damage and cellular senescence in mammals⁸².Several pathologies associated with altered telomere functions have beenreported as “telomeropathies”⁸⁵.

It has recently been appreciated that mammalian genomes are pervasivelytranscribed and the vast majority of DNA sequences can be found inprimary, often overlapping, transcripts most of which apparently notassociated with coding functions³. These non-coding RNAs (ncRNAs) mayremain associated with chromatin, and some aggregate in subnuclearstructures such as speckles and paraspeckles⁴. An unsuspected increasingnumber of these ncRNA transcripts have been shown to be evolutionarilyconserved among related species^(5,6) and play a variety of relevantcellular functions by regulating the localization and the activity ofproteins and/or providing structural support for cellular andsub-cellular structures and controlling chromatin-modification^(4,8) andenhancer-like functions⁹. These activities may be exerted despiteestimated very low levels of expression, few molecules per cell, forsome of these RNA molecules^(10,11,12,13). Some ncRNAs may be processedby ribonucleases of the RNA interference (RNAi) pathway, giving rise toshort double-stranded RNA products that participate in various cellularfunctions. The RNAi pathway is a conserved machinery, whose componentsare thought to have evolved to preserve genome integrity from theattacks of viruses and mobile genetic elements¹⁴. It involves differenttypes of short double-stranded RNA molecules including small interferingRNAs (siRNAs), microRNAs, repeat-associated small interfering RNAs(rasiRNAs), Piwi-interacting RNAs (piRNAs)¹⁵ and QDE-2 interacting RNAs(qiRNA) in Neurospora crassa ¹⁶. It is commonly thought that onlymicroRNA maturation is dependent on both DROSHA and DICER endonucleases,two RNase type III enzymes that process hairpin structures to generatedouble-stranded microRNAs¹⁷. In mammals, microRNAs modulate geneexpression usually by their ability to regulate mRNA translation andstability and have been involved in several processes such as cell fatedetermination, transformation, proliferation and cell death¹⁸. piRNAsand qiRNAs have been implicated in genome stability maintenance¹⁶ and afamily of microRNAs (miR-34) has been shown to act downstream of p53¹⁹.It is presently unknown whether any RNAs have any direct role in thecontrol of DDR activation at sites of DNA damage.

US2006105384 is focused on a technique for detecting and diagnosingdisease conditions, as well as health conditions due to exposure toenvironmental conditions by detecting and identifying DNA or RNA damagemarkers. This technique is based on measurement of free levels ofnucleotide excision products resulting from DNA or RNA damage. TheDDRNAs of the instant invention are not nucleotide excision products.

JP2009171895 concerns a method for analyzing the function of anon-coding RNA (ncRNA) existing in a nucleus by destroying the ncRNA byintroducing an antisense oligo-molecule containing substantially thesame sequence as a sequence complementary to a single-stranded region inthe secondary structure of the target ncRNA to a cell nucleus anddestroying the RNA molecule.

WO2012/013821 relates to the field of cancer, particularly cancerswherein p53 tumour suppression function is lost or impaired. It is shownherein that Dicer is a synthetic lethal partner of p53, allowing theselective targeting and killing of cancer cells. The effects of Dicer onsurvival on cancer cells are mediated through the miR17-92 cluster andinhibition of members of this miRNA cluster is an attractive treatmentstrategy in cancer. Most particularly, these findings are of importancein the field of retinoblastoma.

WO2011/157294 relates to compositions comprising an inhibitor of apolynucleotide, said polynucleotide to be inhibited being capable ofdecreasing or suppressing expression of Dicer or a biologically activederivative thereof for use in treating or preventing cancer, metastasis,heart failure, cardiac remodelling, dilated cardiomyopathy, autoimmunediseases, or diseases or disorders related thereto. Furthermore, thepresent invention also relates to methods of treating or preventingcancer, metastasis, heart failure, cardiac remodelling, dilatedcardiomyopathy, autoimmune diseases, or diseases or disorders relatedthereto. DDRNAs are not mentioned nor the impact of Dicer modulation onDNA damage related events and DDR modulation.

WO2009/102225 relates to compositions and methods for cancer diagnosis,research and therapy, including but not limited to, cancer markers. Inparticular, the present invention relates to ncRNAs as diagnosticmarkers and clinical targets for prostate, lung, breast and pancreaticcancer.

US2012289581 relates to long non-coding RNAs (lncRNAs) and methods ofusing them diagnostically and therapeutically for treatment of cancer,stem cell therapy, or regenerative medicine are disclosed. Inparticular, the invention relates to lncRNAs that play roles inregulation of genes involved in cell proliferation, differentiation, andapoptosis. Such lncRNAs can be used as biomarkers to monitor cellproliferation and differentiation during cancer progression or tissueregeneration. One of the identified lncRNAs, referred to as PANDA (aP21-Associated NcRNA, DNA damage Activated), inhibits the expression ofapoptotic genes normally activated by the transcription factor NF-YA.Inhibitors of PANDA sensitize cancerous cells to chemotherapy and can beused in combination with chemotherapeutic agents for treatment ofcancer.

Limmer K et al. (2013) used a Molecular Force Assay (MFA) to measure theactivity of Dicer. As a model system, they used an RNA sequence thatforms an aptamer-binding site for paromomycin, a 615-daltonaminoglycoside. They have shown that Dicer activity is modulated as afunction of concentration and incubation time: the addition ofparomomycin leads to a decrease of Dicer activity according to theamount of ligand. The measured dissociation constant of paromomycin toits aptamer was found to agree well with literature values. The parallelformat of the MFA allows a large-scale search and analysis for ligandsfor any RNA sequence.

Wei et al, (2012) reports the existence in plants and in a human cancercell line of small RNAs, named diRNAs, generated in proximity to DNA DSBsites⁸¹. The authors show that genetic inactivation of Dicer-like RNAendonucleases results in a specific defect in DNA repair by homologousrecombination. Authors observe some correlation between diRNAsaccumulation and DNA repair by homologous recombination, and proposethat diRNAs control DNA repair. However there is no support by the datashown in the article by Wei et al. to such hypothesis. As a matter offact there is no evidence that diRNA play a biologically active role inthe process of DNA repair. Prior art data in Wei et al are not incontrast with diRNAs being generated following the degradation of a RNAtranscript spanning the DSB site.

In addition, it is not demonstrated in the Wei et al article that theproposed effect of the inactivation of Dicer-like genes and DNA repairis not indirect, possibly mediated by canonical RNA interferencemechanisms. Although the authors show that the abundance of few DNArepair factors is not affected, it is not demonstrated that other DNArepair factors, not tested by the authors, are unaffected and nottargeted by RNA interference mechanisms and thus potentially making anindirect impact on DNA repair.

Finally, correlation is not always maintained and at least in plants theauthors show cases in which diRNAs are decreased (FIG. 3a , mutants RDR2and RDR6) and DNA repair is unaltered (FIG. 3b ).

DDRNA of the instant invention have been characterized for distinctfunctions: DDRNAs control DDR signaling, whereas diRNA of Wei et al arenot shown to have any role in DDR signaling: Wei et al show no evidenceof altered DDR activation, as detected by nuclear DDR foci formation orof DDR proteins activation, for instance by phosphorylation, or ofaltered DNA damage checkpoint functions or modulation of cellularsenescence. Thus there is no demonstrated overlap between theirfunctions.

In cultured Drososphila cells, Michalik et al.⁷⁹, showed that thetransfection of a linearized plasmid leads to the generation of short(21 nt) RNAs with the sequence of the plasmid DNA ends. The small RNAsin this system are produced by active transcription of plasmid genes inthe vicinity of the break. The function proposed for them was therepression of the marker gene encoded by the plasmid. Inactivation ofsome of the factors involved in the RNA interference pathway relievesthe observed repression. Such effect has been interpreted as RNAinterference activity of the short RNAs acting as endo-siRNAs. A causalrelation between the production of short RNA and DDR activation or DNArepair is lacking in this study. This set of observation support thenotion that small RNA are produced at DNA ends in cultured Drosophilacells, but it does not provide a function of this novel RNA molecule inthe DNA damage response pathway.

SUMMARY OF THE INVENTION

DICER (Gene ID: 23405; Official Symbol: DICER1 Name: dicer 1,ribonuclease type III [Homo sapiens] Other Aliases: DCR1, Dicer, HERNA,KIAA0928, MNG1; Other Designations: Dicer1, Dcr-1 homolog; K12H4.8-LIKE;dicer 1, double-stranded RNA25 specific endoribonuclease;endoribonuclease Dicer; helicase MOI; helicase with RNAse motif;helicase-moi, Chromosome: 14; Location: 14q32.13, Annotation: Chromosome14, NC 000014.8 (95552565 . . . 95623759, complement), MIM: 606241, NCBIversion May 4, 2012) and DROSHA (Gene ID: 29102; Official Symbol: DROSHAName: drosha, ribonuclease type III [Homo sapiens], Other Aliases:ETOHI2, HSA242976, RANSE3L, 30 RN3, RNASE3L, RNASEN; Other Designations:RNase III; drosha, double-stranded RNA-specific endoribonuclease;nuclear RNase III Drosha; p241; protein Drosha; putative protein p241which interacts with transcription factor Sp1; putative ribonucleaseIII; ribonuclease 3; ribonuclease III, nuclear; ribonuclease type III,nuclear; Chromosome: 5; Location: 5p13.3, Annotation: Chromosome 5,NC_000005.9 (31400601 . . . 31532282, complement), MIM: 608828, NCBIversion May 4, 2012) are crucial ribonucleases involved in RNAinterference (RNAi). Components of RNAi are thought to have evolved topreserve genome stability from the attacks of viruses and mobile geneticelements. RNA products generated by DICER and DROSHA are involved inchromatin assembly in Schizosaccharomyces pombe, gene silencing andcancer. The DNA damage response (DDR) is a signaling pathway thatarrests the proliferation of cells undergoing genotoxic events topreserve genome stability. So far, RNAi and DDR signaling pathways havenot been demonstrated to directly interact. Here the authors show thatoncogene-induced senescent cells, cells thus bearing oncogene-inducedDNA damage and consequent DDR activation, require DICER and DROSHA tomaintain DDR activation and cell-cycle arrest. DICER and DROSHA are alsonecessary to activate DDR upon exogenous DNA damage, and DDR checkpointfunctions depend on the ribonuclease activity of DICER. DICER isrequired for irradiation-induced DDR activation in vivo in zebrafish. Inan in vitro cellular system, DDR foci stability is sensitive to RNase Atreatment, and DICER- and DROSHA-dependent small RNA products arerequired to restore DDR foci in RNase A-treated cells. Study of DDRactivation at a DNA double-strand break within a unique and traceableexogenous integrated locus reveals that DDR focus formation requireslocus-specific RNA molecules. The authors provide evidence through RNAsequencing that short or small RNAs, that the authors call DDRNAs,originate at the locus and carry the sequence of the damaged locus. Whenchemically synthesized or generated in vitro by DICER cleavage oftranscripts spanning the locus, DDRNAs promote DDR activation at the DNAdamage site in RNase A-treated cells also in the absence of othermammalian RNAs. All together, the authors' results reveal anunanticipated direct role of short or small RNAs (DDRNAs) in the controlof DDR activation at sites of DNA damage.

DDRNAs act differently from microRNAs and canonical RNAi mechanismsbecause:

-   -   DDRNAs act without the need for any other cellular RNA (see the        results obtained with gel-extracted RNA and synthetic RNAs in        RNAse A-treated cells experiments).    -   DDRNAs can have a sequence (LAC or TET repeats) that has no        endogenous cellular transcripts match and still be biological        active    -   DDRNAs can act fast (in minutes) at room temperature in cells        inhibited for transcription and translation (see the results        obtained in RNAse A-treated cells experiments).    -   Inactivation of GW proteins (effectors of canonical miRNA) does        not affect DDR foci. It is therefore an object of the present        invention a method to inhibit the DNA damage response in a cell        damaged in at least one sequence specific genomic locus        comprising the step of:        a) inhibiting the function of small RNAs (DDRNAs), said small        RNAs being generated by processing by DICER and/or DROSHA of a        RNA transcript synthesized upon transcription of the damaged        genomic locus, or impairing the production thereof.

Preferably the method further comprising the step of:

b) exposing said cell to a DNA damaging treatment.

Preferably the DNA damaging treatment belongs to the group of:radiotherapy, chemotherapy (i.e. hydroxyurea treatment, bleomycinetreatment), a treatment that impairs DNA repair or any genotoxictreatment.

It is another object of the invention a method for sensitizing a celldamaged in at least one sequence specific genomic locus to the effect ofa DNA damaging treatment, comprising the step of:

a) inhibiting the function of small RNAs (DDRNAs), said small RNAs beinggenerated by processing by DICER and/or DROSHA of a RNA transcriptsynthesized upon transcription of the damaged genomic locus, orimpairing the production thereof,andb) exposing said cell to an effective amount of the DNA damagingtreatment,wherein step a) and step b) are performed in any order.

Preferably the DNA damaging treatment is a radiotherapy. Stillpreferably the radiotherapy is any ionizing radiation.

In a preferred embodiment the cell is damaged in at least one sequencespecific genomic locus by a genotoxic event.

Preferably the genotoxic event belongs to the group of: celltransformation, cellular senescence, oncogene activation, DNAreplication stress, reactive oxygen species, ionizing radiation,chemotherapeutic agents (i.e. comprising but not limiting to bleomycin),telomere shortening, damaged telomere, recombination including V(D)Jrecombination at the immunoglobulins and T cell receptor locus, viralintegration in the genome, viral infection and replication, bacterialinfection.

In a preferred embodiment the step of inhibiting the function of saidsmall RNAs (DDRNAs) is performed by a sequence specific inhibitormolecule.

Preferably the sequence specific inhibitor molecule is a sequencespecific oligonucleotide. Still preferably the sequence specificinhibitor oligonucleotide is a LNA molecule.

In a preferred embodiment the step of impairing the production of saidsmall RNAs (DDRNAs) is performed by inhibiting the cleavage and/orhelicase activity of DICER and/or DROSHA.

Preferably the inhibitor of the cleavage and/or helicase activity ofDICER and/or DROSHA is a specific siRNA.

In a preferred embodiment the cell is a mammalian cell. Preferably ahuman cell. Yet preferably the cell carries a sequence specific DNAdamaged genomic locus.

Still preferably a pre-cancerous cell, a cancer cell, a senescent cell,a cell with damaged telomeres or a viral infected cell. Preferably, thesenescent cell has critically short and/or damaged and/or dysfunctionaltelomeres.

It is a further object of the invention an inhibitor of small RNAs(DDRNAs), said small RNAs being generated by processing by DICER and/orDROSHA of a RNA transcript synthesized upon transcription of a sequencespecific damaged genomic locus for medical use.

Preferably the inhibitor is for use in the treatment of a conditioninduced by the sequence specific damaged genomic locus.

Preferably the condition is cancer and/or aging and/or a viralinfection. Preferably aging is associated with critically short and/ordamaged and/or dysfunctional telomeres.

In a preferred embodiment the inhibitor is a sequence specific inhibitormolecule. Preferably said sequence specific inhibitor molecule is asequence specific oligonucleotide. Still preferably said sequencespecific inhibitor oligonucleotide is a LNA molecule.

Preferably the inhibitor is an inhibitor of DICER and/or DROSHA. Stillpreferably the inhibitor is a siRNA.

It is a further object of the invention a pharmaceutical compositioncomprising the inhibitor as defined above. The pharmaceuticalcomposition comprises carriers, diluents and/or excipients. Thecomposition may be administered by parenteral, oral, intravenous,intranasal, intramuscular route or any suitable route. Thepharmaceutical composition may be administered in any effective amountto elicit the desired therapeutic effect. The composition may be in anyforms: solution, tablet, ointment etc.

It is a further object of the invention a method to detect the presenceof damage to DNA in a sequence specific genomic locus in a cellcomprising the steps of:

a) detecting the presence of small RNAs (DDRNAs), said small RNAs beinggenerated by processing by DICER and/or DROSHA of a RNA transcriptsynthesized upon transcription of the damaged genomic locus in saidcell;b) comparing the result to a control cell with undamaged DNA genomiclocus.

It is a further object of the invention a method to identify the genomiclocation of a damage to DNA in a sequence-specific genomic locus in acell comprising the steps of:

a) isolating and/or purifying small RNAs (DDRNAs) from a sample, saidsmall RNAs being generated by processing by DICER and/or DROSHA of a RNAtranscript synthesized upon transcription of the damaged genomic locusin said cell;b) sequencing said isolated and/or purified small RNAs (DDRNAs).

It is a further object of the invention a method to quantify the DNAdamage in a specific genomic locus in a cell comprising the steps of:

a) measuring the amount of small RNAs (DDRNAs), said small RNAs beinggenerated by processing by DICER and/or DROSHA of a RNA transcriptsynthesized upon transcription of the damaged genomic locus in saidcell;b) comparing the result to a proper control.

It is a further object of the invention a method for the diagnosisand/or prognosis of a condition associated with and/or induced by thegeneration of DNA damage in at least one sequence specific genomic locuscomprising:

a) measuring the amount of small RNAs, said small RNAs being generatedby processing by DICER and/or DROSHA of a RNA transcript synthesizedupon transcription of the damaged genomic locus in said cell;b) comparing the result to a proper control.

Preferably the condition associated with and/or induced by thegeneration of DNA damage in at least one sequence specific genomic locusis selected from the group consisting of: cancer, aging, viralinfection. Still preferably aging is associated with critically shortand/or damaged and/or dysfunctional telomeres.

It is a further object of the invention a method for monitoring theefficacy of therapy directed to a condition associated with and/orinduced by the generation of DNA damage in at least one sequencespecific genomic locus in a subject comprising:

a) measuring the amount of small RNAs (DDRNAs), said small RNAs beinggenerated by processing by DICER and/or DROSHA of a RNA transcriptsynthesized upon transcription of the damaged genomic locus in saidcell;b) comparing the result to a proper control.

Preferably the condition associated with and/or induced by thegeneration of DNA damage in at least one sequence specific genomic locusis selected from the group consisting of: cancer, aging, viralinfection. Still preferably aging is associated with damaged telomeres.

In the method for monitoring the efficacy of therapy, the proper controlmay be an untreated cell, a healthy cell or a cell at various time pointduring the therapy.

It is a further object of the invention a method of screening for anagent able to inhibit small RNAs (DDRNAs), said small RNAs beinggenerated by processing by DICER and/or DROSHA of a RNA transcriptsynthesized upon transcription of a damaged genomic locus in a cellcomprising the step of measuring the amount of said small RNAs uponexposure of the cell to said agent, and comparing to a proper control.

In the method of screening for an agent able to inhibit small RNAs, theproper control may be a cell treated with a reference compound or anon-treated cell.

In the present invention, DDRNAs are small RNAs, with the potential toform double-stranded pairs, that are generated by processing by DICERand/or DROSHA of a sequence specific RNA transcript synthesized upontranscription of a damaged DNA locus. DDRNAs are small RNAs of a lengthbetween 10 and 50 nucleotides. For example of a length between 17 and 32nucleotides. For example of a length between 20 and 25 nucleotides. Forexample of a length between 22 and 23 nucleotides.

Said DDRNAs function by favoring the sequence-specific accumulation ofDDR factors at specific sites of DNA damage and promote DDR signaling(i.e. comprising but not limiting to protein phosphorylation events).

A critically short telomere is a telomere able to engage the DDRmachinery due to its critical short length. A damaged telomere is atelomere carrying a DNA lesion able to engage the DDR machinery. Adysfunctional telomere is a telomere that due to its altered proteinand/or nucleic acid structure engages the DDR machinery

In the present invention an oncogenic stress may be a genotoxic stress(i.e. comprising but not limiting to DNA lesions, impaired DNAreplication forks progression) due to oncogene activation,amplification, gain of function mutation, increased levels and activity.A cell carrying a DNA damage is a cell whose DNA damage is notexogenously induced (i.e. a cell comprising but not limiting tocritically short telomere and damaged telomere, oncogenic stress,oxidative DNA damage).

Aging is associated with telomeric DNA damage and DDR activation^(2,84.)

Genotoxic treatments commonly used in cancer therapy are treatmentsassociated with the generation of DNA damage (i.e. comprising but notlimiting to radiotherapy and chemotherapy).

A radiotherapy is a therapy based on the exposure to ionizing radiation.

An effective dose of ionizing radiation is a dose of ionizing radiationable to generate the desired outcome. The skilled person in the artusing common routine techniques knows how to determine such dose.

A senescent cell is a cell retaining persistent DDR activation (usuallyfollowing oncogenic stress and/or telomere shortening/DNA damage).

The presence of DNA damage is concluded by the presence of said shortRNAs (DDRNAs); in the absence of said DDRNAs, it is concluded that cellsdo not have DNA damage.

In qualitative analysis, control cell may be a non-damaged cell or ahealthy cell. The analysis may be carried out by quantitative ReverseTranscriptase-PCR, northern blot hybridization, next generationsequencing (Illumina etc), ion torrent technology or by any other meansavailable, appropriated and known to the skilled person in the art. Itmay be performed on a cell or the blood or other biological fluids. Itcan also be performed in tissue lysates.

The quantity of said short RNAs (DDRNAs) is proportional to DNA damage,higher quantities of said RNAs indicate larger amount of DNA damage.

In quantitative analysis, the control may be a non-damaged cell or ahealthy cell. The analysis may be carried out by qRT-PCR, northern blothybridization, next generation sequencing (Illumina etc), ion torrenttechnology or by any other means available, appropriated and known tothe skilled person in the art. It may be performed on a cell or theblood or other biological fluids. It can also be performed in tissuelysates.

In the present invention, “inhibiting DICER and/or DROSHA” means:

-   -   1. Inhibiting the enzymatic (nuclease and/or helicase) activity        of DICER and/or DROSHA by means of a small molecule compound        and/or;    -   2. Inhibiting the synthesis of DICER and/or DROSHA by RNA        interference means and/or;    -   3. Destabilizing the proteins DICER and/or DROSHA by targeting        by various means protein cofactors that bind and are necessary        for DICER and/or DROSHA activities and/or;    -   4. Inhibiting DICER and/or DROSHA activity by the expression of        DICER and/or DROSHA alleles with dominant negative functions        and/or;    -   5. Inhibiting DICER and/or DROSHA by targeting the genomic loci        responsible for their synthesis.

An inhibitor of DICER and/or DROSHA is able to have at least one of theabove activities. In the present invention “inhibiting small RNAs, saidsmall RNAs being generated by processing by DICER and/or DROSHA of a RNAtranscript synthesized upon transcription of the damaged genomic locus”means:

-   1. preventing DDRNAs biogenesis and/or processing by inhibiting    DICER and/or DROSHA as described above and/or-   2. preventing DDRNAs synthesis by preventing the transcription of    the longer RNA precursor eventually cleaved by DICER and/or DROSHA    and/or-   3. preventing DDRNAs proper localization in the cell to prevent    their processing and/or-   4. preventing DDRNAs proper localization in the cell to prevent    their functions and/or-   5. preventing DDRNAs accumulation by increasing their degradation    rates and/or-   6. preventing DDRNAs functions by the use of a sequence specific    inhibitory oligonucleotide able to avidly and specifically bind to    DDRNAs. These oligonucleotides comprise but are not limiting to    locked nucleic acids (LNA), phosphorothioate modified    oligonucleotides, 2′-O-methoxyethyl modified oligonucleotides, 2′    O-Methyl modified oligonucleotides, methylphosphonates, morpholino    oligonucleotides, LNA-DNA-LNA gapmer oligonucleotides, Chimeric    2′-O-methyl RNA-DNA gapmer, N3′-P5′ Phosphoroamidate,    2′-fluoro-arabino nucleic acid, Phosphoroamidate Morpholino,    Cyclohexene nucleic acid, Tricyclo-DNA, Peptide nucleic acid,    Unlocked nucleic acid, Hexitol nucleic acid, Boranophosphate    oligonucleotides, Phosphoroamidate oligonucleotides and/or    oligonucleotides expressed by plasmid-encoded genes delivered by    different means (comprising but not limited to plasmid transfection,    viral infection) and/or-   7. preventing DDRNAs functions by modifying them by means i.e. of    methylation, and/or-   8. preventing any modification of DDRNAs (such as phosphorylation,    methylation etc etc. . . . ) that may be necessary for their    function, stability or localization.

A DICER and/or DROSHA inhibitor is an agent or molecule able to displayat least one DICER and/or DROSHA inhibiting function as described above(inhibiting the enzymatic activity of DICER and/or DROSHA, inhibitingthe synthesis of DICER and/or DROSHA, destabilizing the proteins DICERand/or DROSHA, inhibiting DICER and/or DROSHA activity by the expressionof DICER and/or DROSHA alleles with dominant negative functions and/or;targeting the genomic loci responsible for DICER and/or DROSHAsynthesis).

An inhibitor of DDRNAs is an agent or molecule able to display at leastone DICER and/or DROSHA inhibiting function as described above(preventing their synthesis, preventing their proper localization in thecell to prevent their processing and/or functions, preventing theiraccumulation, preventing their functions, preventing them to act as theywould and/or preventing any modification of DDRNAs).

The invention will be now described by way of non-limiting examplesreferring to the following figures.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F|DICER or DROSHA inactivation impairs DDR foci stability andformation in OIS and in irradiated cells. FIG. 1A DICER and DROSHAknockdown by siRNA pools in OIS cells impairs DDR foci maintenance asdetected by 53BP1, pATM, pS/TQ, γH2AX immunostaining. FIG. 1B Histogramsshow quantification of the percentage of cells positive for 53BP1, pATM,pS/TQ and γH2AX or H3K9me3, a marker of SAHF formation. FIG. 1C DICER orDROSHA knockdown by siRNA pools in WI38 human fibroblasts impairs pATM,pS/TQ, MDC1, but not γH2AX, foci assembly. Cells were irradiated (10Gy)and fixed 7 h later. FIG. 1D Histograms show percentage of WI38 cellspositive for pATM, pS/TQ, MDC1 and γH2AX foci. FIG. 1E pATM, pS/TQ andMDC1, but not γH2AX, foci formation is impaired inDICER^(exon5)-hypomorphic cells. Cells were irradiated (2Gy) and fixed2h later. FIG. 1F Histograms show the percentage of cells positive forDDR foci. Error bars indicate s.e.m. (n≥3). Differences (*) arestatistically significant (p-value<0.001).

FIGS. 2A-2D)|DICER-inactivated cells have impaired G1/S and G2/Mcheckpoint. FIG. 2A DICER, DROSHA or 53BP1 knockdown by siRNA pools inWI38 impairs irradiation-induced G1/S checkpoint. siGFP was used ascontrol. Cells were irradiated (10Gy) and labeled with BrdU for 7 hoursbefore fixation. Histograms show the percentage of BrdU-positive cellsin not-irradiated (−) and in irradiated (+) cells. FIG. 2BDICER^(exon5)-hypomorphic cells have impaired G1/S checkpoint. Cellswere irradiated (2Gy) and labeled with BrdU for 2 hours before fixation.Histograms show the percentage of BrdU-positive cells in not-irradiated(−) and in irradiated (+) cells. Wild-type DICER-flag cDNA expressionrestores the G1/S checkpoint while cDNAs of DICER endonuclease mutantsDICER44ab-flag and DICER110ab-flag do not. Error bars indicate s.e.m.(n=3). Differences (*) are statistically significant (p-value<0.01).FIG. 2C DICER knocked-down cells have impaired G2/M checkpoint. FACSprofiles of HEK293 cells transfected with an shRNA against DICER and p53at 12, 24 and 36 hours post irradiation (5Gy). shGFP is used as control.FIG. 2D The table shows the percentage of cells in G1, S and G2 phase ofthe cell cycle at different time points post IR.

FIGS. 3A-3D|Irradiation induces pATM and γH2AX nuclear accumulation incontrol but not in Dicer1 morpholino-injected zebrafish embryos. FIG. 3AImages illustrating the location of the sections from the head of 3 dayspost fertilization (dpf) WT (not injected) and Dicer1-morpholinoinjected zebrafish larvae stained for pATM and γH2AX before and afterirradiation (12 Gy). Sections were stained with DAPI (blue) and pATM orγH2AX antibody (green). FIG. 3B Immunoblot analysis of pATM and γH2AXaccumulation in extracts from not irradiated and irradiated wild-typeembryos or Dicer1 morpholino-injected embryos. Total ATM and histone H3were used as loading control. FIG. 3C Schematic drawing of thetransplantation procedure. GFP-positive Dicer1 morpholino transplantedcells, integrated in various locations in the host, show reduced γH2AXsignals following IR. FIG. 3D Schematic drawing of the reversetransplantation procedure: control cells from embryos injected with mRNAencoding for GFP were transplanted into Dicer1 morpholino injectedembryos. Dicer1-expressing cells display γH2AX signals, while thesurrounding Dicer1 morpholino-injected cells do not.

FIGS. 4A-4E|Irradiation-induced DDR foci are sensitive to RNase Atreatment and are restored by short and DICER RNA products. FIG. 4AIrradiated HeLa cells (2 Gy) were treated with PBS (−) or RNase A (+)and probed for 53BP1, pATM, pS/TQ, MDC1 and γH2AX foci. 53BP1, pATM,pS/TQ and MDC1, but not γH2AX, foci are strongly reduced upon RNase Atreatment. FIG. 4B Histograms report the percentage of cells positivefor DDR foci. FIG. 4C Addition of gel-purified RNA in the size range of20-35 nt allows DDR foci reformation in RNase A-treated cells. RNAs wereseparated according to their size by acrylamide gel electrophoresis andgel extracted. 100, 50 or 20 ng of gel extracted total RNA and 50 ng ofRNA extracted from each gel fraction (>100 nt, 35-100 nt and 20-35 nt)were used for RNA reconstitution post RNase treatment in HeLa cells.Error bars indicate s.e.m. (n=2). Differences are statisticallysignificant (*p-value<0.01). FIG. 4D Irradiation-induced 53BP1, pS/TQand pATM foci are restored in RNase A-treated cells when incubated withRNA of wild-type (WT RNA) RKO cells but not with RNA ofDICER^(exon5)-hypomorphic (DICER^(exon5) RNA) RKO cells. tRNA was usedas control. γH2AX foci were not affected. FIG. 4E Histograms report thepercentage of cells positive for DDR foci. Error bars indicate s.e.m.(n≥4). Differences are statistically significant (*p-value<0.001).

FIGS. 5A-5E|Site-specific DDR focus formation is RNase A-sensitive andcan be restored by locus-specific RNA in a MRE11-RAD50-NBS1complex-dependent manner. FIG. 5A NIH2/4 mouse cells experiencing I-SceI-induced DSB next to a Lac-operator array (LacO array) display a 53BP1(green) and γH2AX (magenta) focus colocalizing with the Cherry-Lac focus(red). 53BP1, but not γH2AX focus, is sensitive to RNase A and it isrestored by incubation with total RNA. FIG. 5B Histograms show thepercentage of cells in which 53BP1 and Cherry-Lac foci co-localize.Addition of 50, 200, 800 ng of RNA purified from NIH2/4 rescues 53BP1foci formation in a dose-dependent manner. FIG. 5C Incubation of RNaseA-treated cells with RNA purified from NIH2/4 expressing I-Sce Irestores 53BP1 focus at the I-Sce I induced cut site, while RNA fromNIH3T3 parental cells expressing I-Sce I does not. FIG. 5D RNA fromNIH2/4 cells, or parental one, was used in RNase A-treated NIH2/4 cellsto test 53BP1 focus reformation in the presence of the MRN inhibitormirin. Cells were pretreated with 100 μM mirin for 15 minutes at roomtemperature before RNA addition and mirin was kept at the sameconcentration during incubation with RNA. Both 53BP1 and pATM focireformation is inhibited by the presence of mirin. Histograms report thepercentage of cells positive for DDR foci. Error bars indicate s.e.m.(n≥3). Differences are statistically significant (*p-value<0.05). FIG.5E RNA from NIH2/4 cells, or parental one, was used in RNase A-treatedNIH2/4 cells to test pATM focus reformation in the presence of the MRNinhibitor mirin. Cells were pretreated with 100 μM mirin for 15 minutesat room temperature before RNA addition and mirin was kept at the sameconcentration during incubation with RNA. Both 53BP1 and pATM focireformation is inhibited by the presence of mirin. Histograms report thepercentage of cells positive for DDR foci. Error bars indicate s.e.m.(n≥3). Differences are statistically significant (*p-value<0.05).

FIGS. 6A-6D|Chemically synthesized locus-specific RNAs and in vitrogenerated DICER RNA products promote DDR focus formation at the DNAdamage site in RNase A-treated cells. FIG. 6A A pool of chemicallysynthesized oligonucleotides was tested to restore DDR focus formationin RNase A-treated NIH2/4 cells. Mixed with a constant amount of RNAfrom parental cells (800 ng), a wide range of concentrations (1 ng/μl-1fg/μl, ten-fold dilution steps) of locus-specific or control (GFP) RNAswas used. Locus-specific synthetic RNAs (down to a concentration of 100fg/μl), but not control ones, allow site-specific DDR activation. FIG.6B Short RNAs generated by recombinant DICER processing of RNA generatedin vitro by transcription of a DNA fragment carrying the central portionof the integrated locus, or a control one of similar length, were testedto restore DDR focus formation at the DNA damage site in RNase A-treatedNIH2/4 cells. RNAs were tested at the concentration of 1 ng/μl mixedwith 800 ng of RNA from parental cells. Locus-specific DICER RNAproducts, but not control ones, allow site-specific DDR activation. FIG.6C The fraction of 22-23 nt vs total short RNAs at the locus decreasesin DICER and DROSHA knockdown samples both in uncut and cut conditions.In DICER knockdown samples the decrease is statistically significant (inthe uncut samples p=4.8e−7, in the cut samples p=0.029). The fraction of22-23 nt vs total short RNAs at the locus increases in the wildtype uponcutting (p=0.02). The statistical significance was calculated by fittinga negative binomial model to the short RNA count data and performing alikelihood ratio test, keeping the fraction of 22-23 nt vs total RNAs atthe locus fixed across conditions under the null hypothesis and allowingit to vary between conditions under the alternative hypothesis. FIG. 6DThe distribution of nucleotides at the 5′ and the 3′ end of RNAsequences from the damaged locus is depicted in the sequence logo,showing that 82.9% of sequences start with adenine or uracil and that48.6% of sequences end with guanine.

FIGS. 7A-7G|DICER or DROSHA inactivation in OIS cells allows escape fromsenescence and cell-cycle progression. FIG. 7A DICER, DROSHA knockdownby siRNA pools in OIS cells were evaluated by QRT-PCR. ATM knockdown bysiRNA pool was evaluated by immunofluorescence. FIG. 7B DICER or DROSHAknockdown in OIS cells by siRNA increases BrdU incorporation rates.Histograms show the percentage of BrdU-positive cells. siGFP was used ascontrol. Error bars indicate s.e.m. (n≥3). Differences are statisticallysignificant (p-value<0.001). FIG. 7C DICER-, DROSHA- and DDR-inactivatedOIS cells by transfection with siRNA pools, re-express MCM2, a marker ofchromosomal DNA replication. Error bars indicate s.e.m. (n≥3).Differences are statistically significant (p-value<0.001). FIG. 7FDICER-, DROSHA- and DDR-inactivated OIS cells by transfection with siRNApools, re-express pH3, a marker of entry into mitosis. Error barsindicate s.e.m. (n≥3). Differences are statistically significant(p-value<0.001). FIG. 7D DICER and DROSHA knockdown was evaluated byQRT-PCR and FIG. 7G DICER and DROSHA knockdown was evaluated by QRT-PCRFIG. 7E 53BP1 knockdown is evaluated by immunofluorescence.

FIGS. 8A-8E|Different concentrations and individual siRNA against DICERor DROSHA in OIS cells reproducibly allow escape from senescence. FIG.8A 200 nM and 20 nM concentration of siRNA pools against DICER or DROSHAin OIS cells allow escape from senescence. Error bars indicate s.e.m.(n≥3). Differences are statistically significant (p-value<0.05).Knockdown was evaluated by QRT-PCR. FIG. 8B 200 nM and 20 nMconcentration of siRNA pools against DICER or DROSHA in OIS cells allowescape from senescence. Error bars indicate s.e.m. (n≥3). Differencesare statistically significant (p-value<0.05). Knockdown was evaluated byQRT-PCR. FIG. 8C 200 nM and 20 nM concentrations of siRNA pools againstDICER or DROSHA in OIS cells allow escape from senescence. Error barsindicate s.e.m. (n≥3). Differences are statistically significant(p-value<0.05). Knockdown was evaluated by QRT-PCR. FIG. 8D siRNA poolsagainst DICER or DROSHA used in OIS cells were deconvolved and siRNAswere tested individually and they reproducibly allow escape fromsenescence. Error bars indicate s.e.m. (n≥3). Differences arestatistically significant (p-value<0.05). Knockdown was evaluated byQRT-PCR. FIG. 8E siRNA pools against DICER or DROSHA used in OIS cellswere deconvolved and siRNAs were tested individually and theyreproducibly allow escape from senescence. Error bars indicate s.e.m.(n≥3). Differences are statistically significant (p-value<0.05).Knockdown was evaluated by QRT-PCR.

FIGS. 9A-9F|DICER or DROSHA inactivation in OIS cells does not alterSAHF maintenance and does not decrease DDR protein levels but impairstheir activation over a range of siRNA concentrations. FIG. 9A DICER orDROSHA were inactivated by transfection with siRNA pool in OIS cells.Cells were stained for H3K9me3 SAHF marker and for 53BP1. siGFPtransfected cells are used as control. DICER or DROSHA inactivationaffects 53BP1 foci without altering SAHF stability. FIG. 9B Immunoblotanalysis of H3K9me3 in DICER or DROSHA inactivated OIS cells. Total H3is used as loading control. FIG. 9C Immunoblot analysis of 53BP1, ATMand H2AX in DICER or DROSHA inactivated OIS cells. siGFP transfectedcells are used as control. Vinculin is used as loading control. FIG. 9DDifferent concentrations (10 fold difference) of siRNA pools againstDICER or DROSHA in OIS cells impair DDR foci detection. Error barsindicate s.e.m. (n≥3). Differences are statistically significant(p-value<0.05). Knockdown was evaluated by QRT-PCR. FIG. 9E Differentconcentrations (10 fold difference) of siRNA pools against DICER orDROSHA in OIS cells impair DDR foci detection. Error bars indicates.e.m. (n≥3). Differences are statistically significant (p-value<0.05).Knockdown was evaluated by QRT-PCR. FIG. 9F Different concentrations (10fold difference) of siRNA pools against DICER or DROSHA in OIS cellsimpair DDR foci detection. Error bars indicate s.e.m. (n≥3). Differencesare statistically significant (p-value<0.05). Knockdown was evaluated byQRT-PCR.

FIGS. 10A and 10B|DICER or DROSHA, but not GW182, inactivation in OIScells impairs DDR foci formation. FIG. 10A DICER, DROSHA or GW182/TNRC6Awere inactivated by siRNA pool in OIS cells. DICER-, DROSHA- orGW182/TNRC6A-inactivated cells were stained for 53BP1, pATM and pS/TQmarkers of activated DDR. GW182/TNRC6A inactivation has no detectableeffect on DDR. Differences for DICER and DROSHA, but not GW182, arestatistically significant (p-value<0.005). Error bars indicate s.e.m.(n≥3). FIG. 10B Knockdown was evaluated by QRT-PCR.

FIGS. 11A and 11B|Simultaneous inactivation of TNRC6A/GW182, TNRC6B andTNC6C in OIS cells does not affect DDR foci formation while DROSHAinactivation does. FIG. 11A OIS cells inactivated by individual siRNAfor DROSHA or TNRC6A, B and C simultaneously (with two different sets ofsiRNAs: pool #1 and #2) were stained for 53BP1, pATM and pS/TQ markersof activated DDR. TNRC6A, B and C simultaneous inactivation with eithersiRNA pools #1 or #2 has no detectable effect on DDR. Differences forDROSHA, but not TNRC6A, B and C, are statistically significant(p-value<0.05). Error bars indicate s.e.m. (n≥3). FIG. 11B Knockdown wasevaluated by QRT-PCR.

FIGS. 12A-12G|DICER or DROSHA inactivation in HNF impairs IR-induced DDRfoci formation. FIGS. 12A, 12B Efficiency of DICER or DROSHA knockdownin WI38 human fibroblasts was evaluated by immunostaining FIG. 12A andQRT-PCR FIG. 12B, respectively. FIG. 12C Immunoblot analysis of ATM,53BP1 and H2AX proteins expression levels in DICER- orDROSHA-inactivated WI38. siGFP transfected cells are used as control.Vinculin is used as loading control. FIGS. 12D, 12E, 12F, and 12G siRNApools against DICER or DROSHA were deconvolved and siRNAs were usedindividually. They all reduce DDR foci formation. 53BP1, but not γH2AXis reduced FIG. 12D and pATM and pS/TQ are reduced, FIG. 12E.Differences are statistically significant (p-value<0.005). Error barsindicate s.e.m. (n≥3). FIG. 12F Knockdown was evaluated by QRT-PCR. FIG.12G Knockdown was evaluated by QRT-PCR.

FIGS. 13A-13D|53BP1 foci formation is delayed upon DICER or DROSHAknockdown and impaired DDR foci formation is rescued by wild type butnot mutant DICER. FIG. 13A 53BP1-foci formation is impaired 10 minutespost IR (10 Gy) in WI38 cells knocked-down for DICER or DROSHA by siRNApools. Histograms show the percentage of WI38 cells positive for 53BP1foci. Differences (*) are statistically significant (p-value<0.05). FIG.13B Expression of siRNA-resistant wt DICER, but not a mutant allelelacking endonuclease activity, rescues DDR foci formation defect inDICER knocked-down cells. FIG. 13C 53BP1 foci formation was studied 10′after IR (10 Gy), pATM and MDC1 1 hour afterward. Differences (*) arestatistically significant (p-value<0.001). Error bars indicate s.e.m.(n=3). FIG. 13D Knockdown of endogenous DICER by 3′UTR siRNA wasevaluated by QRT-PCR.

FIGS. 14A-14C|DICER or DROSHA, but not GW182, inactivation in HNFimpairs DDR foci formation. FIG. 14A DICER, DROSHA or GW182/TNRC6A wasinactivated in Wi38 cells by siRNA pools, cells were irradiated (2Gy)and stained for 53BP1 10′ later, for γH2AX. FIG. 14B DICER, DROSHA orGW182/TNRC6A was inactivated in Wi38 cells by siRNA pools, cells wereirradiated (2Gy) and stained for pATM and pS/TQ markers of activatedDDR, 1 hour post IR. GW182/TNRC6A inactivation has no detectable effecton DDR. Differences for DICER and DROSHA, but not GW182, arestatistically significant (p-value<0.001). Error bars indicate s.e.m.(n≥3). FIG. 14C Knockdown was evaluated by QRT-PCR.

FIGS. 15A-15D|DICER inactivation and TNRC6 A, B and C, simultaneousinactivation in HeLa cells is associated with similar levels of theco-transfected RFP-miR126 sensor mRNA but DDR foci are impaired only inDICER-inactivated cells FIG. 15A HeLa cells were cotransfected withRFP-miR126 sensor mRNA and siRNA against DICER or TNRC6A, B and C in apool. Cells were irradiated (2Gy) and stained for 53BP1 10′ later. FIG.15B Both DICER and GW182-like proteins inactivation results in theupregulation of the miR126 sensitive RFP reporter but only DICERinactivation has detectable effect on DDR. Differences for DICER but notTNRC6 A, B and C, are statistically significant (p-value<0.05). Errorbars indicate s.e.m. (n≥3). FIG. 15C Knockdown was evaluated by QRT-PCR.FIG. 15D The relative miRNA quantity of DICER and TNRC6A, B, and C inHeLa cells transfected with siRNA against DICER or TNRC6A, B and C isdepicted.

FIGS. 16A-16C|DDR factors are expressed but 53BP1 foci formation isdelayed in DICER^(exon5)-hypomorphic RKO cells. FIG. 16A Immunoblotanalysis of ATM, MDC1, 53BP1 and H2AX in wild-type (WT) andDICER^(exon5)-hypomorphic RKO cells. Vinculin and tubulin are used asloading control. FIG. 16B Irradiated DICER^(exon5)-hypomorphic RKO cellshave delayed 53BP1-foci formation. Images show 53BP1-foci at 10, 30 and90 minutes post IR (2 Gy) in wild-type (WT) andDICER^(exon5)-hypomorphic RKO cells. FIG. 16C Histograms show thepercentage of RKO cells positive for 53BP1 foci. Error bars indicates.e.m. (n=3). Differences (*) are statistically significant(p-value<0.05).

FIGS. 17A and 17B|Impaired DDR foci formation inDICER^(exon5)-hypomorphic cells is rescued by wild type but not mutantDICER. FIG. 17A Expression of wt DICER but not a mutant allele lackingendonuclease activity, restores DDR foci formation defect inDICER^(exon5)-hypomorphic RKO cells. 53BP1 foci formation was studied10′ after IR (2 Gy), pATM and pS/TQ 1 hour afterward. FIG. 17BHistograms show the percentage of cells positive for the indicated DDRfoci. Differences (*) are statistically significant (p-value<0.001).Error bars indicate s.e.m. (n≥3).

FIGS. 18A-18C|ATM activation by autophosphorylation is impaired in DICERand DROSHA-inactivated cells. FIG. 18A ATM activation following IR (10Gy) is impaired in DICER and DROSHA knocked-down WI38 human fibroblastsas detected by immunoblot analysis for pATM. siGFP transfected cells areused as a positive control for ATM activation. Total ATM is unaffectedas shown by vinculin. DICER knockdown was evaluated by immunoblotanalysis. FIG. 18B DROSHA knockdown in WI38 cells was evaluated byQRT-PCR. FIG. 18C ATM activation is impaired in irradiated (2 Gy)DICER^(exon5)-hypomorphic RKO cells. Total ATM and vinculin are used asloading control.

FIGS. 19A-19G|DICER or DROSHA knockdown impairs G1/S checkpoint. DICER,DROSHA and 53BP1 knockdowns by siRNA pools in WI38 cells were monitoredby QRT-PCR FIG. 19A, immunoblot FIG. 19B and immunofluorescence FIG.19C, respectively. FIG. 19D siRNA pools against DICER or DROSHA used inWI38 cells were deconvolved and siRNAs were used individually and theyreproducibly impair G1/S checkpoint activation. Histogram shows thepercentage of BrdU positive cells before (black bar) and after IR (graybar) upon normalization on the percentage of BrdU-positive cells beforeIR for each cell line. Error bars indicate s.e.m. (n=3). FIG. 19EKnockdown was evaluated by QRT-PCR. FIG. 19F DICER-inactivated MRC-5cells have impaired G1/S checkpoint post IR (10 Gy). siGFP is used ascontrol. FIG. 19G DICER and 53BP1 knockdowns efficiency in MRC-5 wasmonitored by immunofluorescence. Error bars indicate s.e.m. (n=3).Differences (*) are statistically significant (p-value<0.05).

FIGS. 20A-20G|Loss of G1/S and G2/M checkpoint activation in DICERknocked-down cells. FIG. 20A DICER-flag cDNA transfection into HCT116DICER^(exon5)-hypomorphic cells restores a proficient G1/S checkpointpost IR (2 Gy). Histograms show the percentage of BrdU-positive cells.Error bars indicate s.e.m. (n=3). Differences (*) are statisticallysignificant (p-value<0.01). FIG. 20B Immunoblot analysis againstflag-epitope shows the expression of DICER mutants (DICER-flag,DICER110ab-flag and DICER44ab-flag) in RKO cells. FIG. 20C DICERknockdown by shRNA in HEK293 cells was monitored by QRT-PCR. FIG. 20DDICER-inactivated HEK293 cells have impaired G2/M checkpoint as detectedby pH3 immunostaining of mitotic cells in not irradiated cells and 24 hpost IR (5 Gy). Histograms show the percentage of pH3 positive cells incontrol and DICER-inactivated cells. Error bars indicate s.d. (n=3).Differences are statistically significant by student's t-test(p-value<0.05). FIG. 20E DICER knocked-down cells have an impaired G2/Mcheckpoint that can be restored upon transfection of siRNA-resistantDICER. The table shows the percentage of cells in G1, S and G2 phase ofthe cell cycle at different time points post IR. FIG. 20F FACS profilesof HEK293 cells transfected with the indicated combinations of siRNA andvectors (EV stands for empty vector), 36 hours post IR (5Gy). FIG. 20GEndogenous DICER (En DICER) knockdown and exogenous DICER (Exo DICER)overexpression were evaluated by QRT-PCR.

FIGS. 21A-21D|Dicer1 morpholino-injected zebrafish embryos downregulatethe expression of miRNAs. FIG. 21A 72 hours post fertilization (hpf)larvae injected with Red Fluorescent Protein (RFP) miR126 sensor mRNA (1larva on the left) or RFP miR126 sensor mRNA together with Dicer1morpholino (3 larvae on the right). Note the jaw defects, small eye anddelayed yolk re-absorption indicative of developmental delays in theDicer1 morpholino-injected embryos. The same embryos visualized byepifluorescence show an increase in RFP expression in Dicer1 morpholinoinjected embryos. FIG. 21B Specificity of miR126 sensor. Upper panelsshow the expression levels of RFP miR126 sensor mRNA and GFP mRNA in thecontrol, uninjected embryos. Lower panel shows the effects of Dicer1morpholino injection on the expression of RFP miR126 sensor (increasedin the absence of mature miR126) and GFP (unchanged). FIG. 21C miRNAexpression of 48 and 72 hours post fertilization embryos injected or notwith Dicer1 morpholino were analyzed by real-time PCR. Data areexpressed as relative expression level of Dicer1 morpholino-injectedembryos compared to the control embryos not injected and are the mean oftwo independent pools of embryo performed in duplicate. FIG. 21D Tableof raw CT values.

FIGS. 22A-22D|RNase A treatment degrades both mRNAs and microRNAs,leaves DDR protein levels unaltered but compromises their activation.FIG. 22A QRT PCR analysis of β-actin mRNA and miR-21 in mock and RNaseA-treated cells. Error bars indicate s.d. (n=3). Differences arestatistically significant by Student's t-test (p-value<0.05). FIG. 22BQRT PCR analysis of 53BP1 and ATM mRNA in RNase A-treated cells. Errorbars indicate s.d. (n=3). Differences are statistically significant byStudent's t-test (p-value<0.05). FIG. 22C RNase A treatment does notaffect DDR (ATM, 53BP1, MDC1) protein stability. Lamin A/C is used asloading control. FIG. 22D RNase A affects 53BP1 and MDC1 but not □H2AXin the same focus. Irradiated HeLa cells were treated with PBS or RNaseFIG. 22A 53BP1 foci (green) and MDC1 foci (red) are affected by RNase Atreatment while γH2AX (magenta) foci in the same cell are not.

FIGS. 23A and 23B|α-amanitin inhibits spontaneous DDR foci reformationfollowing RNase A treatment. FIG. 23A Irradiation-induced (2 Gy) 53BP1foci are α-amanitin sensitive. HeLa cells were irradiated and incubatedwith PBS (−) or RNase A (+). Afterwards cells were incubated with theRNase A inhibitor RNaseOUT, with or without α-amanitin for 10 or 45minutes. Incubation with α-amanitin prevents 53BP1-foci reformation.FIG. 23B Histogram shows the percentage of cells positive for 53BP1foci. Error bars indicate s.e.m. (n=3). Differences (*) arestatistically significant (p-value<0.01).

FIGS. 24A-24C|Ppo I-induced DDR foci are RNase A sensitive and reformupon RNA addition. HeLa cells were transfected with the active form ofPpo I, a restriction enzyme, treated with RNase A and incubated withtRNA, as a control, or their own RNA. Histograms show the percentage ofcells positive for the indicated DDR markers. Error bars indicate s.e.m.(n=3). Differences (*) are statistically significant (p-value<0.005).FIG. 24A shows the percentage of cells positive for 53BP1. FIG. 24Bshows the percentage of cells positive for pATM. FIG. 24C shows thepercentage of cells positive for pS/TQ.

FIGS. 25A-25C|Short RNAs promote DDR foci reformation at the DNA damagesite FIG. 25A Relative enrichment of miR-21 RNA compared to β-actin mRNAquantity evaluated by QRT-PCR in total RNA and short RNA-enrichedfractions. FIG. 25B Histograms show the percentage of cells positive for53BP1, pATM and pS/TQ foci in irradiated HeLa cells, RNase A-treated,and cells incubated with 200 ng of total RNA or a proportional volume ofshort RNA-enriched (<200 nt) fraction. tRNA (200 ng) was used ascontrol. FIG. 25C Irradiation-induced 53BP1 (green) and pATM (red) fociare restored in RNase A-treated cells by incubation with total and shortRNAs-enriched fraction. tRNA was used as control.

FIGS. 26A and 26B|Irradiation-induced DDR foci are restored in RNaseA-treated cells by incubation with RNAs extracted from gel in the 20-35nt range. FIG. 26A Irradiated cells were RNase A treated and incubatedwith equal amounts (50 ng) of RNA extracted from polyacrilamide gel asshown in FIG. 4C Images show 53BP1 staining in cells incubated with theindicated RNAs. FIG. 26B QRT PCR analysis of RNU19 (200 nt), RNU44 (61nt) and mir-21 (22 nt) in the indicated RNA fractions extracted fromgel. Error bars indicate s.d. (n=3). Differences are statisticallysignificant by student's t-test (p-value<0.005).

FIGS. 27A and 27B|RNA extracted from DICER^(exon5)-hypomorphic cellstransfected with wild type but not mutant DICER allows DDR focireformation in RNase A-treated cells. FIG. 27A Irradiated cells wereRNase A-treated and incubated with RNA extracted from the indicatedcells. RNA from wild type cells or DICER^(exon5)-hypomorphic cellstransfected with wild type DICER allows 53BP1 foci reformation, whileRNA from untransfected DICER^(exon5)-hypomorphic cells or the same cellstransfected with mutant DICER do not. FIG. 27B Histograms show thepercentage of cells positive for the indicated DDR markers. Error barsindicate s.e.m. (n=3). Differences (*) are statistically significant(p-value<0.005).

FIGS. 28A-28E|DICER and DROSHA RNA products are required for DDR focireformation FIG. 28A RNA extracted from wild-type HCT116 and DLD-1 celllines, but not that extracted from DICER^(exon5)-hypomorphic HCT116 andDLD-1 cell lines, rescues 53BP1 foci. Histograms show the percentage ofcells positive for 53BP1 foci. Error bars indicate s.e.m. (n=3).Differences are statistically significant (p-value<0.01). FIG. 28B RNAextracted from shDICER-transfected (or GFP as control) HEK293 cells doesnot restore irradiation-induced 53BP1 foci in RNase A-treated HeLacells. tRNA is used as negative control. FIG. 28C Histograms report thepercentage of cells positive for 53BP1 foci. FIG. 28D Immunoblottingshows DICER knockdown efficiency. FIG. 28E Histograms show thepercentage of cells positive for 53BP1, pATM and pS/TQ foci inirradiated HeLa cells after RNase A treatment and incubation with RNApurified from siGFP and siDROSHA transfected cells. tRNA was used ascontrol.

FIGS. 29A and 29B|MRN complex involvement in DDR foci reformation afterRNase treatment. FIG. 29A MRN complex recruitment to the I-Sce I-inducedDSB is sensitive to RNase A treatment. 53BP1, pATM and MRE11 foci, butnot γH2AX, are lost in RNase A treated NIH2/4 cells. Histograms show thepercentage of cells in which 53BP1, pATM, MRE11 or γH2AX focicolocalizing with the Cherry-Lac focus. FIG. 29B Mirin impairs pATMactivation on the I-Sce I-induced DSB. Histogram shows the percentage ofcells in which pATM focus colocalize with the Cherry-Lac focus. Errorbars indicate s.e.m. (n≥3). Differences are statistically significant(*p-value<0.05).

FIGS. 30A-30H|Identification of biologically active locus-specificmolecules. Chemically synthesized locus-specific RNAs and in vitrogenerated DICER RNA products promote DDR focus formation at the DNAdamage site in RNase A-treated cells. FIG. 30A Nuclear RNA shorter than200 nt was purified and analyzed on the small RNA Bioanalyser kit(Agilent). FIG. 30B Short RNA library was prepared and extracted from 6%polyacrylamide gel (indicated by an arrow at 100 bp). FIG. 30C Theintegrity of the prepared library was checked using the Agilent DNA highsensitivity kit. FIG. 30D Length distribution of tags in the library.FIG. 30E Length distribution of tags in the library mapping to theexogenous integrated locus combining tags from cut and uncut samples.FIG. 30F A pool of chemically synthesized oligonucleotides mapping tothe exogenous locus was tested to restore DDR focus formation in RNaseA-treated NIH2/4 cells. Mixed with a constant amount of tRNA, a widerange of concentrations (0.1 ng/μl, 0.1 pg/μl and 1 fg/μl) oflocus-specific or control (GFP) RNAs, was used. FIG. 30G DICERprocessing was evaluated by running DICER RNA-products on a 3% agarosegel. FIG. 30H Short RNAs cleaved by recombinant DICER processing of RNAgenerated in vitro upon transcription of a DNA fragment carrying thecentral portion of the integrated locus, or a control one of similarlength, were tested to restore DDR focus formation in RNase A-treatedNIH2/4 cells. RNAs were tested at the concentration of 1 ng/μl mixedwith 800 ng of tRNA. Locus-specific DICER RNA products, but not controlones, allow site-specific DDR activation at the DNA damage site.Histograms report the percentage of cells positive for DDR foci.

FIGS. 31A-31I|Library profile and length distribution of selectedsequenced samples. FIG. 31A Bioanalyser profile of <200 nt RNA fromwildtype cut sample. FIG. 31B Short RNA libraries were prepared from 40ng RNA from each sample and run on a 6% PAGE gel. Arrow shows the 100 bplibrary band of interest. FIG. 31C Wildtype cut library profile. Gelextracted libraries were run on Bioanalyser high sensitivity kit.Sequencing was performed on Hi seq Version 3. FIG. 31D Tag lengthdistribution of wildtype uncut. FIG. 31E Tag length distribution ofDicer KD uncut. FIG. 31F Tag length distribution of Drosha KD uncut.FIG. 31G Tag length distribution of wildtype uncut. FIG. 31H Tag lengthdistribution of Dicer KD cut. FIG. 31I Tag length distribution of DroshaKD uncut.

FIGS. 32A and 32B|Dicer and Drosha knockdown downregulates miRNAs. FIG.32A Dicer and Drosha knockdown by shRNA in uncut and cut samples wasevaluated by QRT-PCR. FIG. 32B Reads mapping to the miRNA databasemiRBase release 18 were normalized with the number of reads of spike ineach library. Normalized miRNAs in Dicer and Drosha knockdown sampleswere compared with wildtype samples before and after cut (as labeled).Statistical significance was calculated using the Wilcoxon signed-ranktest. The authors find that miRNAs are significantly lower expressed inthe Dicer and Drosha knockdown sample compared to the wildtype sample inboth cut and uncut conditions (Dicer knockdown uncut vs wildtype uncutp=1.544e−263; Drosha knockdown uncut vs wildtype uncut p=3.843e−279;Dicer knockdown cut vs wildtype cut p=8.911e−84; Drosha knockdown cut vswildtype cut p=1.172e−275).

FIGS. 33A-33D|Features of short RNAs arising from the locus. Length oftags arising from the locus before and after cut. Y-axis shows number oftags from the locus and X-axis depicts tag lengths in nucleotides. Thebulk of short RNAs in wildtype samples before and after cut are in the22-23 nt size range. Among knockdown samples, Dicer knockdown shows abroader tag length distribution. FIG. 33A depicts the length of tagsarising from the locus before and after cutting. FIG. 33B depicts thelength of tags arising from the locus before and after cutting withshDICER. FIG. 33C depicts the length of tags arising from the locusbefore and after cutting with shDROSHA. FIG. 33D 22-23 nt percentage ofthe locus is significantly different from the same ratio of non miRNAgenomic loci. Fractions of 22-23 nt vs total short RNAs at non miRNAgenomic loci with at least 50 reads are shown in histograms with thevertical axis depicting their frequency. In each sample, the verticalline depicts the ratio of 22-23 nt RNAs to the total at the locus. Thep-value was calculated by summing the area (indicated in red) to theright of this line. The authors find that the fraction of 22-23 nt vstotal short RNAs at the locus studied is significantly higher than thefraction of 22-23 nt tags at non miRNA genomic loci in both uncut(p=0.049) and cut (p=0.022) conditions.

FIGS. 34A-34C|Sequence-specific inhibitory oligonucleotides (i.e. LNAs)transfection impairs DDR at the locus in cut cells. The scheme FIG. 34Ashows the TET-I-SceI-LAC locus, the DDRNAs generated upon cut (greyline), and the LNAs used (black dotted line). Cells were co-transfectedwith Cherry-Lac and I-Sce I-restriction endonuclease expressing vectorstogether with different sets (as in the legend in the figure) of LNA(200 μM) with the potential to anneal to DDRNAs arising from the locusupon I-SceI-induced cleavage or a control LNA matching telomeresequence. 24 h post transfection cells were scored for DDR markers atthe Lac array. Histograms show the percentage of cells positive for theDDR markers analysed: γH2AX FIG. 34B is not affected, whereas 53BP1 FIG.34C accumulation at the locus is significantly reduced upon all sets oftransfected LNAs. Around 100 cells from three independent experimentswere scored. Error bars indicate s.e.m. Statistical significance wascalculated by Chi-squared test compared to control LNA (sample 2).*p-value<0.05, **p-value<0.01, ***p-value<0.005.

FIGS. 35A and 35B|DDR activation (53BP1 focus maintenance) at uncappedtelomeres is RNA-dependent. TRF2^(flox/flox) MEFs (Lazzerini Denchi andde Lange, Nature 2007) were treated with 4-hydroxytamoxifen to inducecre-mediated TRF2 knockout and generate uncapped telomeres. 48 hourslater, cells were permeabilized and treated with RNase A or BSA, as acontrol. FIG. 35A Representative images show that γH2AX foci are stable,while 53BP1 foci disassemble upon RNase A treatment. FIG. 35BQuantification of γH2AX and 53BP1 foci in RNase A and BSA treated cells.For the quantifications shown around 150 cells from two independentexperiments were scored. Error bars indicate s.e.m. ***p-value<0.001.

FIGS. 36A-36C|Sequence-specific inhibitory oligonucleotides (i.e. LNAs)transfection suppresses DDR and prevents BrdU reduction followingtelomere-uncapping. T19 fibrosarcoma cell line (van Steensel, Cell 1998)was cultured in absence of doxycycline to induce expression of adominant negative allele of TRF2 fused to flag. Induced cells arevisualized by Flag immunostaining. Induced (Flag+) and uninduced (Flag−)cells were transfected with LNA molecules (200 nM) matching the sense(LNA 6), the antisense (LNA 5) telomeric sequence, and an unrelated (LNA3, Cntrl) sequence at day 13 from induction. FIG. 36A 53BP1foci-positive cells were scored at the indicated time points posttransfection. LNA 5 and 6 cause a decrease in DDR-positive cells, todifferent extent, compared to control LNA in induced cells, while nodifference was observed in uninduced cells. For the quantificationsshown, around 30-100 cells were scored for each time point FIGS. 36B,36C Induced cells were incubated with BrdU for 16 hours and scored forBrdU incorporation 3 days following LNA transfection. LNA 5 and 6transfected Flag+ cells show a significant increase in the percentage ofBrdU-positive cells, compared to the Cntrl LNA FIG. 36B, while nodifference is observed in Flag− cells FIG. 36C. For the quantificationsshown around 150 cells from three FIG. 36B or two FIG. 36C independentexperiments were scored. Error bars indicate s.e.m. *p-value<0.05,***p-value<0.001.

DETAILED DESCRIPTION OF THE INVENTION Methods

Cultured Cells.

Early passage BJ cells, WI38 and MRC-5 (The American Type CultureCollection, ATCC) were grown under standard tissue culture conditions(37° C., 5% CO₂) in MEM supplemented with 10% fetal bovine serum, 1%L-glutamine, 1% non-essential aminoacids, 1% Na Pyruvate. HeLa, Phoenixecotrophic and HEK293T cell lines were grown under standard tissueculture conditions (37° C., 5% CO₂) in DMEM, supplemented with 10% fetalbovine serum, 1% glutamine, 1% penicillin/streptomycin. RKO, HCT116 andDLD1 colon cancer cell lines²⁵ were cultured in Mc'Coy 5A medium+10%fetal calf serum, 1% penicillin/streptomycin. NIH2/4³⁵ where grown inDMEM, supplemented with 10% fetal bovine serum, 1% glutamine,gentamicine (40 μg/ml), and hygromycin (400 μg/ml).

H-RasV12 overexpressing senescent BJ cells were generated as in²⁰. BrdUincorporation assays were carried at least a week after cultures hadfully entered the senescent state, as determined by ceasedproliferation, DDR activation, SAHF formation, and senescence-associatedβ-galactosidase expression. Ionizing radiation (IR) was induced by ahigh-voltage X-rays generator tube (Faxitron X-Ray Corporation). Ingeneral, cultured cells were exposed to 2 Grays for the foci formationassay. The authors used 5 Grays for the G2/M checkpoint assays and 10Grays for the G1/S checkpoint assays.

Cherry-Lac and I-Sce I-restriction endonuclease expressing vector weretransfected by lipofectamine 2000 (Invitrogen) in a ratio of 3:1. 16 hpost transfection around 70% of the cells were scored positive for DDRmarkers at the Lac array. For generation of Dicer and Droshaknocked-down NIH2/4 cells were infected with Lentiviral particlescarrying pLKO.1, shDicer or shDrosha vectors. After 48 hours cells weresuperinfected with Adeno Empty Vector or Adeno I-Sce I [Anglana et al.Nucl Ac Res 1999]. Nuclei were isolated the day after the adenoviralinfection.

Transient expression of ER-I-Ppol endonucleases in HeLa cells wascarried out by Lipofectamine 2000 transfection and 16 hours latertamoxifen (0.1 μM) was added to culture medium to induce the activationof the endonuclease. 4 hours later cells were fixed for immunostainingor used for RNA extraction. Cherry-Lac transfected (mock) cells wereused as control in these experiments.

Cultured Cells and LNA Transfection (for the Experiments in FIG. 34).

NIH2/4 cells where grown in DMEM, supplemented with 10% fetal bovineserum, 1% glutamine, gentamicine (40 mg/ml), and hygromycin (400 mg/ml).Cherry-Lac and I-Sce I-restriction endonuclease expressing vectors weretransfected with Lipofectamine 2000 (Invitrogen) with a 3:1 ratio. LNAwere first boiled at 90° C. for 5 minutes and quickly chilled at 4° C.for 5 minutes and then added in different combinations to the Cherry-Lacand I-Sce I transfection mix, at the final concentration of 200 pM. 24 hpost transfection cells were scored for DDR markers at the Lac array.

Cultured Cells (for the Experiments in FIGS. 35-36).

T19 fibrosarcoma cells (van Steensel, Cell 1998) were grown in DMEMsupplemented with 10% fetal bovine serum, 1% glutamine and doxycycline(100 ng/ml). For induction, cells were grown without doxycycline for atleast 7-8 days. CRE-ER TRF2^(flox/flox) MEFs (Lazzerini Denchi and deLange, Nature 2007) were grown in DMEM supplemented with 10% fetalbovine serum and 1% glutamine. For induction, cells were grown inpresence of 4-hydroxytamoxifen (600 nM) for 48 hours. For BrdUincorporation, cells were labeled with 10 μg/ml bromodeoxyuridine (BrdU,Sigma) for 16 hours and incorporation was evaluated byimmunofluorescence after DNA denaturation.

Antibodies.

Mouse anti-γH2AX, anti-H3K9me3, rabbit polyclonal anti-PH3 (UpstateBiotechnology); anti-pS/TQ (Cell Signaling Technology); anti-H2AX,anti-H3 and anti DICER (13D6) (Abcam); rabbit polyclonal anti-53BP1(Novus Biological) and mouse monoclonal anti-53BP1 (a gift from ThanosHalazonetis); anti-MRE11 (a gift from S. Jackson); anti pH3, anti-BrdU(Becton Dickinson); rabbit polyclonal anti-MCM2 (a gift of MarineMelixetian); anti MRE11 rabbit polyclonal raised against recombinantMRE11; anti-pATM (Rockland); mouse monoclonal anti-ATM and anti-MDC1(SIGMA); anti-vinculin (clone hVIN-1), anti-β-tubulin (clone AA2) andanti-Flag M2 monoclonal antibodies (SIGMA).

Indirect Immunofluorescence.

Cells were grown on poly-D-lysinated coverslips (poly-D-lysine was usedat 50 μg/ml final concentration) and plated (15-20×10³ cells/cover) oneday before staining. DDR and BrdU staining was performed as in²⁰. Cellswere fixed in 4% paraformaldehyde or methanol:acetone 1:1. NIH2/4 mousecells were fixed by 4% paraformaldehyde as in³⁵. Images were acquiredusing a wide field Olympus Biosystems Microscope BX71 and the analySISor the MetaMorph software (Soft Imaging System GmbH). Comparativeimmunofluorescence analyses were performed in parallel with identicalacquisition parameters; at least 100 cells were screened for eachantigen. Cells with more than 2 DDR foci were scored positive. Fociintensity quantifications were performed using Cell Profiler software2.0. Confocal sections were obtained with a Leica TCS SP2 AOBS confocallaser microscope by sequential scanning.

Immunofluorescence (for the Experiments in FIGS. 35-36).

Cells were fixed with 1:1 methanol/acetone solution for 2 minutes atroom temperature, or 4% paraformaldehyde for 10 minutes at roomtemperature. After blocking, cells were stained with primary antibodiesfor 1 h at room temperature, washed and incubated with conjugatedsecondary antibodies for 40 minutes at RT. Nuclei were stained with DAPI(1 μg/ml).

Plasmids.

Flag-DICER, Flag-DICER44ab and Flag-DICER110ab were a kind gift of R.Shiekhattar. pLKO.1 shDICER expressing vector was a kind gift of WC.Hahn. Short hairpin sequence for DICER is: CCG GCC ACA CAT CTT CAA GACTTA ACT CGA GTT AAG TCT TGA AGA TGT GTG GTT TTT G (SEQ ID NO:1).pRETROSUPER shp53 as in²⁰. Short hairpin sequence for p53 was: AGT AGATTA CCA CTG GAG TCT T (SEQ ID NO:2). Cherry-Lac-repressor and I-SceI-restriction endonuclease expressing vectors were kind gifts of E.Soutoglou³⁵. ER-I-Ppo I-restriction endonuclease expressing vector was akind gift of Michael Kastan³³. shRNA against mouse Dicer and Droshaexpressing vectors were a kind gift of W. C. Hahn. shRNA for mouseDicer: CCG GGC CTC ACT TGA CCT GAA GTA TCT CGA GAT ACT TCA GGTCAA GTGAGG CTT TTT (SEQ ID NO:3). shRNA for mouse Drosha: CCG GCC TGG AAT ATGTCC ACA CTT TCT CGA GAA AGT GTG GAC ATA TTC CAG GTT TTT G (SEQ ID NO:4).

siRNA.

The DHARMACON siGENOME SMARTpool siRNA oligonucleotide sequences forhuman 53BP1, ATM, DICER, DROSHA were:

53BP1: (SEQ ID NO: 5) GAG AGC AGA UGA UCC UUU A; (SEQ ID NO: 6)GGA CAA GUC UCU CAG CUA U; (SEQ ID NO: 7) GAU AUC AGC UUA GAC AAU U;(SEQ ID NO: 8) GGA CAG AAC CCG CAG AUU U. ATM: (SEQ ID NO: 9)GAA UGU UGC UUU CUG AAU U; (SEQ ID NO: 10) AGA CAG AAU UCC CAA AUA A;(SEQ ID NO: 11) UAU AUC ACC UGU UUG UUA G; (SEQ ID NO: 12)AGG AGG AGC UUG GGC CUU U. DICER: (SEQ ID NO: 13)UAA AGU AGC UGG AAU GAU G; (SEQ ID NO: 14) GGA AGA GGC UGA CUA UGA A;(SEQ ID NO: 15) GAA UAU CGA UCC UAU GUU C; (SEQ ID NO: 16)GAU CCU AUG UUC AAU CUA A. DROSHA: (SEQ ID NO: 17)CAA CAU AGA CUA CAC GAU U; (SEQ ID NO: 18) CCA ACU CCC UCG AGG AUU A;(SEQ ID NO: 19) GGC CAA CUG UUA UAG AAU A; (SEQ ID NO: 20)GAG UAG GCU UCG UGA CUU A.

The DHARMACON siGENOME si RNA sequences for Human TNRC6A, B and C were:

GW182/TNRC6A: (SEQ ID NO: 21) GAA AUG CUC UGG UCC GCU A; (SEQ ID NO: 22)GCC UAA AUA UUG GUG AUU A. TNRC6B: (SEQ ID NO: 23)GCA CUG CCC UGA UCC GAU A; (SEQ ID NO: 24) GGA AUU AAG UCG UCG UCA U.TNRC6C: (SEQ ID NO: 25) CUA UUA ACC UCG CCA AUU A; (SEQ ID NO: 26)GGU AAG UCC UCC AUU GAU G. siRNA against human DICER 3′ UTR:(SEQ ID NO: 27) CCG UGA AAG UUU AAC GUU U. siRNA against GFP:(SEQ ID NO: 28) AAC ACU UGU CAC UAC UUU CUC. siRNA against Luciferase:(SEQ ID NO: 29) CAU UCU AUC CUC UAG AGG AUG dTdT; (SEQ ID NO: 30)dTdT GUA AGA UAG GAG AUC UCC UAC.

siRNAs were transfected by Oligofectamine (Invitrogen) at a finalconcentration of 200 nM in OIS cells and 100 nM in HNF. In the siRNAtitration experiment we transfected OIS cells in parallel with 20 nM and200 nM siRNA oligos. For siRNA transfection with deconvolved siRNAoligos the authors used 50 nM for smart pools and 12.5 nM fordeconvolved siRNAs.

Real-Time Quantitative PCR (RT-QPCR).

Total RNA was isolated from cells using TRIzol (Invitrogen) or RNAeasykit (Qiagen) according to the manufacturer's instructions, and treatedwith DNAse before reverse transcription. For microRNA isolation theauthors used mirVana™ miRNA Isolation Kit (Ambion). cDNA was generatedusing the Superscript II Reverse Transcriptase (Invitrogen). cDNA wasused as template in TaqMan® Gene Expression Assays (Applied Biosystems)for the evaluation of DICER (Assay ID: Hs00998580_ml) and DROSHA (AssayID: Hs01095030_ml) mRNA levels. TaqMan® MicroRNA Assays (AppliedBiosystems) were used for the evaluation of mature miR-21 and rnu44 andrnu19 expression levels (Assay ID: 000397, 001094 and 001003). 18S orβ-actin was used as a control gene for normalization. miR21 and rnu44enrichment in the small RNA-enriched fraction was evaluated as the ratiobetween PCR cycles (ct) for miR-21 or rnu44 and for β-actin mRNA afternormalization to the same ratio in total RNA fraction. Real-timequantitative PCR reactions were performed on an Applied Biosystems ABIPrism 7900HT Sequence Detection System or on a Roche LightCycler 480Sequence Detection System. The reactions were prepared using SyBR Greenreaction mix from Roche. Ribosomal protein P0 (RPP0) was used as a humanand mouse control gene for normalization.

Primer sequences for real-time quantitative PCR were:

RPPO: (Forward) (SEQ ID NO: 31) TTCATTGTGGGAGCAGAC, (Reverse)(SEQ ID NO: 32) CAGCAGTTTCTCCAGAGC; human endogenous DICER: (Forward)(SEQ ID NO: 33) AGCAACACAGAGATCTCAAACATT, (Reverse) (SEQ ID NO: 34)GCAAAGCAGGGCTTTTCAT; human endogenous and overexpressed DICER: (Forward)(SEQ ID NO: 35) TGTTCCAGGAAGACCAGGTT, (Reverse) (SEQ ID NO: 36)ACTATCCCTCAAACACTCTGGAA; human DROSHA: (Forward) (SEQ ID NO: 37)GGCCCGAGAGCCTTTTATAG, (Reverse) (SEQ ID NO: 38) TGCACACGTCTAACTCTTCCAC;human GW182: (Forward) (SEQ ID NO: 39) CAGCCAGTCAGAAAGCAGTG, (Reverse)(SEQ ID NO: 40) TGTGAGTCCAGGATCTGCTACTT; mouse Dicer: (Forward)(SEQ ID NO: 41)  GCAAGGAATGGACTCTGAGC, (Reverse) (SEQ ID NO: 42)GGGGACTTCGATATCCTCTTC; mouse Drosha: (Forward) (SEQ ID NO: 43)CGTCTCTAGAAAGGTCCTACAAGAA, (Reverse) (SEQ ID NO: 44)GGCTCAGGAGCAACTGGTAA.

RNase A Treatment and RNA Complementation Experiments.

Cells were plated on poly-D-lysinated coverslips and irradiated with 2Gy of IR. 1 h after HeLa cells were permeabilized with 2% Tween 20 inPBS for 10 minutes at RT while I-Sce I-transfected NIH2/4 cells werepermeabilized in 0.5% Tween 20 in PBS for 10 minutes at RT. RNase Atreatment was carried out in 1 ml of 1 mg/ml Ribonuclease A from bovinepancreas (Sigma-Aldrich cat n: R5503) in PBS for 25 minutes at roomtemperature. After RNase A digestion, the samples were washed with PBS,treated with 80 units of RNase inhibitor (RNaseOUT Invitrogen 40units/μl) and 2 μg/ml of α-amanitin (SIGMA) for 15 minutes in a totalvolume of 70 μl. For experiments with mirin, NIH2/4 cells were incubatedat this point also with 100 μM mirin (SIGMA) or DMSO for 15 minutes.Then, RNase A-treated cells were incubated with total, small or gelextracted RNA, or the same amount of tRNA, for additional 15 minutes atroom temperature. If using mirin, NIH2/4 cells were incubated with totalRNA in the presence of 100 μM mirin or DMSO for 25 minutes at roomtemperature. Cell were then fixed with 4% paraformaldehyde ormethanol:acetone 1:1.

In complementation experiments with synthetic RNA oligonucleotides,eight RNA oligonucleotides with the potential to form four pairs werechosen among the sequences obtained by deep sequencing that map at theintegrated locus in NIH2/4 cells. Synthetic RNA oligonucleotides weregenerated by SIGMA with a monophosphate modification at the 5′ end.Sequences map to different regions of the integrated locus: two pairsmap to a unique sequence flanking the I-Sce I restriction site (Oligo1+Oligo 2 and Oligos 3+Oligo 4), one to the Lac-operator (Oligo 5+Oligo6) and one to the Tet-repressor repetitive sequences (Oligo 7+Oligo 8).Two paired RNA oligonucleotides with the sequences of GFP were used asnegative control (Oligo GFP 1+Oligo GFP 2). Sequences are reportedbelow.

DDRNA Sequences

Oligo 1: (SEQ ID NO: 45) 5'-AUA ACA AUU UGU GGA AUU CGG CGC-3', oligo 2:(SEQ ID NO: 46) 5'-CGA AUU CCA CAA AUU GUU AUC C-3', oligo 3:(SEQ ID NO: 47) 5'-AUU UGU GGA AUU CGG CGC CUC UAG AGU CGA GG-3',oligo 4: (SEQ ID NO: 48) 5'-CCU CGA CUC UAG AGG CG-3', oligo 5:(SEQ ID NO: 49) 5'-AGC GGA UAA CAA UUU GUG GCC ACA UGU GGA-3', oligo 6:(SEQ ID NO: 50) 5'-UGU GGC CAC AAA UUG UU-3', oligo 7: (SEQ ID NO: 51)5'-ACU CCC UAU CAG UGA UAG AGA AAA GUG AAA GU-3', oligo 8:(SEQ ID NO: 52) 5'-CUU UCA CUU UUC UCU AUC ACU GAU AGG GAG UG-3' GFP 1:(SEQ ID NO: 53) 5'-GUU CAG CGU GUC CGG CGA GUU-3', GFP 2:(SEQ ID NO: 54) 5'-CUC GCC GGA CAC GCU GAA CUU-3'

RNAs were resuspended in 60 mM KCl, 6 mM HEPES-pH 7.5, 0.2 mM MgCl2, atthe stock concentration of 12.5 μM, denatured at 95° C. for 5 minutesand annealed for 10 minutes at room temperature.

DICER RNA products were generated as follows. A 550 bp DNA fragmentcarrying the central portion of the genomic locus studied (three Lacrepeats, the I-Sce I site and two Tet repeats) was flanked by T7promoters at both ends and was used as a template for in vitrotranscription with the TurboScript T7 transcription kit (AMSBIO). The500 nt long RNA obtained was purified and incubated with humanrecombinant DICER enzyme (AMSBIO) to generate 22-23 nt RNAs. RNAproducts were purified, quantified and checked on a polyacrylamide or anagarose gel. As a control, the same procedure was followed with a 700 bpconstruct containing the RFP DNA sequence. Equal amounts of DICERproducts generated in this way were used in complementation experimentin NIH2/4 cells following RNase treatment.

RNaseA Treatment (for the Experiments in FIG. 35).

CRE-ER TRF2^(flox/flox) MEFs (Lazzerini Denchi and de Lange, Nature2007) were induced to generate TRF2 knockout and telomere uncapping. 48hours later cells were permeabilized with 0.6% Tween 20 in PBS for 15min at room temperature. RNase A treatment was carried out in 1 ml of 1mg/ml ribonuclease A from bovine pancreas (Sigma-Aldrich catalogue no.R5503) in PBS for 30 minutes at room temperature.

LNA Transfection (for the Experiments in FIG. 36).

LNA were first boiled at 90° C. for 5 minutes, chilled at 4° C. for 5minutes and transfected with Lipofectamine RNAiMAX (Invitrogen) at thefinal concentration of 200 nM.

Small RNA Preparation.

Total RNA was isolated from cells using TRIzol (Invitrogen) according tothe manufacturer's instructions. To generate small RNA-enriched fractionand small RNA-devoid fraction the authors used mirVana™ microRNAIsolation Kit (Ambion) according to the manufacturer's instructions. ThemirVana microRNA isolation kit employs an organic extraction followed byimmobilization of RNA on glass-fiber (silica-fibers) filters to purifyeither total RNA, or RNA enriched for small species. For total RNAextraction ethanol is added to samples, and they are passed through aFilter Cartridge containing a glass-fiber filter, which immobilizes theRNA. The filter is then washed a few times, and finally the RNA iseluted with a low ionic-strength solution. To isolate RNA that is highlyenriched for small RNA species, ethanol is added to bring the samples to25% ethanol. When this lysate/ethanol mixture is passed through aglass-fiber filter, large RNAs are immobilized, and the small RNAspecies are collected in the filtrate. The ethanol concentration of thefiltrate is then increased to 55%, and it is passed through a secondglass-fiber filter where the small RNAs become immobilized. This RNA iswashed a few times, and eluted in a low ionic strength solution. Usingthis approach consisting of two sequential filtrations with differentethanol concentrations, an RNA fraction highly enriched in RNA species≤200 nt can be obtained^(25,66).

RNA Extraction from Gel.

Total RNA samples were heat-denatured, loaded and resolved on a 15%denaturing acrylamide gel [1×TBE, 7 M urea, 15% acrylamide (29:1acryl:bis-acryl)]. Gel was run for 1 hour at 180 V and stained in GelRedsolution. Gel slices were excised according to the molecular weightmarker, moved to a 2 ml clean tube, smashed and RNA was eluted in 2 mlof ammonium acetate 0.5 M, EDTA 0.1 M in RNase-free water, rockingovernight at 4° C. Tubes were then centrifuged 5 minutes at top speed,the aqueous phase was recovered and RNA was precipitated and resuspendedin RNase free water.

G1/S Checkpoint Assay.

WI38, BJ and MRC-5 cells were irradiated with 10Gy and 1 hour afterwardsincubated with BrdU for 7 h; HCT116 and RKO cells were irradiated 2Gyand incubated with BrdU for 2 h. Cells were fixed with 4%paraformaldehyde and probed for BrdU immunostaining. At least 100 cellsper condition were analyzed.

G2/M Checkpoint Assay.

HEK 293 calcium phosphate transfected cells were irradiated with 5 Gyand allowed to respond to IR-induced DNA damage in a cell cultureincubator for 12, 24 or 36 hours. Then, at these three time points postirradiation, together with not irradiated cells, 1×10⁶ cells werecollected for Fluorescence Activated Cell Sorting (FACS) analysis, fixedin 75% ethanol in PBS, 30 minutes on ice. Afterwards, cells were treated12 hours with 40 μg/ml of RNase A and incubated at least 1 h withpropidium iodide (PI). FACS profiles were obtained by the analysis of atleast 5×10⁵ cells. In the complementation experiments HEK 293 weretransfected using Lipofectamine RNAi Max (Invitrogen) and 48 hours laterirradiated with 5 Gy. Cells were then treated as explained above.

Immunoblotting.

Cells were lysed in sample buffer and 50-100 μg of whole cell lysatewere resolved by SDS-PAGE, transferred to nitrocellulose and probed asin²⁰.

For zebrafish immunoblotting protein analysis, 72 hours postfertilization (hpf) larvae were deyolked in Krebs Ringer's solutioncontaining 1 mM EDTA, 3 mM PMSF and proteases inhibitor (Roche completeprotease inhibitor cocktail). Embryos were then homogenized in SDSsample buffer containing 1 mM EDTA with a pestle, boiled 5 min andcentrifuged 13000 rpm for 1 min. Protein concentration was measured withthe BCA method (Pierce) and proteins (50 μg-900 μg) were loaded in anSDS-12% (for γH2AX and H3) and SDS-6% polyacrylamide gel (for pATM andATM), transferred to a nitrocellulose membrane, and incubated withanti-γH2AX (1:2000, a gift from J. Amatruda⁶⁷), H3 (1:10000, Abcam),pATM (1:1000, Rockland), ATM (1:1000, Sigma). Immunoreactive bands weredetected with horseradish peroxidase-conjugated anti-rabbit oranti-mouse IgG and an ECL detection kit (Pierce, Springfield, Ill.,USA). Protein loading was normalized to equal amounts of total ATM andH3.

Zebrafish Embryo Injection, Cell Transplantation and Staining.

Zebrafish embryos at the stage of 1-2 cells were injected with amorpholino against Dicer1²⁹ diluted in Danieau buffer. The morpholinooligonucleotide was injected at a concentration of 5 ng/nl, and a volumeof 2 nl/embryo. To assess the efficiency of the morpholino to blockmicroRNA maturation, the authors co-injected the morpholino with invitro synthesized mRNA, encoding for red fluorescent protein (RFP) andcarrying 3 binding site for miR126 in the 3′ UTR²⁸. The oligonucleotidescarrying the binding sites for miR126 used for construction ofpCS2:RFPmiR126 sensor are:

(SEQ ID NO: 55) 5'GCATTATTACTCACGGTACGAATAAGGCATTATTACTCACGGTACGAATAAGGCATTATTACTCACGGTACGA 3' and (SEQ ID NO: 56)5'CGTAATAATGAGTGCCATGCTTATTCCGTAATAATGAGTGCCATGCTTATTCCGTAATAATGAGTGCCATGCT 3'.

The construct was verified by sequencing and used to synthesize mRNA invitro using the mMessage Kit (Ambion). Messenger RNA encoding forRFPmiR126 sensor was injected alone or in combination with Dicer1morpholino at a concentration of 10 pg/nl. Dicer morpholino was injectedat a concentration of 5 ng/nl, and a volume of 2 nl/embryo. For celltransplantation experiments, the authors injected donor embryos with amixture of dicer1 morpholino and mRNA encoding for GFP (5 pg/nl).Approximately 20 cells were transplanted from donor embryos at dome (5hpf) stage to uninjected host at the same ng substantially the samesequence as a sequence complementary to a single-stranded region inbelow. Mature miRNA were reverse transcribed to produce 6 different cDNAfor TaqMan® MicroRNA assay (30 ng of total mRNA for each reaction;Applied Biosystems). Real-time PCR reactions based on TaqMan reagentchemistry were performed in duplicate on ABI PRISM® 7900HT FastReal-Time PCR System (Applied Biosystems). The level of miRNA expressionwas measured using CT (threshold cycle). Fold change was generated usingthe equation 2^(−CT).

For immunofluorescence in zebrafish larvae: 72 hpf larvae wereirradiated with 12Gy, fixed in 2% paraformaldehyde for 2 hours at roomtemperature. After equilibration in 10 and 15% sucrose in PBS, larvaewere frozen in OCT compound on coverslips on dry ice. Sections were cutwith a cryostat at a nominal thickness of 14 □m and collected onSuperfrost slides (BDH). Antisera used were zebrafish γH2AX—a kind giftof J. Amatruda⁶⁷—and pATM (Rockland). GFP fluorescence in transplantedembryos was still easily visible in fixed embryos. Images were acquiredwith a confocal (Leica SP2) microscope and 63× oil immersion lens.

RNA Sequencing.

Nuclear RNA shorter than 200 nt was purified using mirVana™ microRNAIsolation Kit. RNA quality was checked on a small RNA chip (Agilent)before library preparation (Supplementary FIG. 23 a). For Illumina hiSeq Version3 sequencing, spike RNA was added to each RNA sample in theRNA:spike ratio of 10,000:1 before library preparation and libraries forIllumina GA IIX were prepared without spike. An improved short RNAlibrary preparation protocol was used to prepare libraries⁶⁸. In brief,adenylated 3′ adapters were ligated to 3′ ends of 3′-OH short RNAs usinga truncated RNA ligase enzyme followed by 5′ adapter ligation to5′-monophosphate ends using RNA ligase enzyme, ensuring specificligation of undegraded short RNAs. cDNA was prepared using a primerspecific to the 3′ adapter in the presence of Dimer eliminator andamplified for 12-15 PCR cycles using a special forward primer targetingthe 5′ adapter containing additional sequence for sequencing and areverse primer targeting the 3′ adapter. The amplified cDNA library wasrun on a 6% polyacrylamide gel and the 100 bp band containing cDNAs upto 33 nt was extracted using standard extraction protocols. Librarieswere sequenced after quality check on a DNA high sensitivity chip(Agilent). Multiplexed barcode sequencing was performed on IlluminaGA-IIX (35 bp Single end reads) and Illumina Hi seq version3 (51 bpsingle end reads). Sequences of all the DDRNAs identified in this studywill be available for free downloading by the time of publication atshort read archive.

Statistical Analyses.

Results are shown as means plus/minus standard error (s.e.m.). p-valuewas calculated by Chi-squared test. QRT-PCR results are shown as meansof a triplicate plus/minus standard deviation (s.d.) and p-value wascalculated by Student's t-test as indicated. * indicates p-value<0.05.

Results in FIGS. 34b and c are shown as means plus standard error(s.e.m.). p-value was calculated by Chi-squared test. * indicatesp-value<0.05, ** indicates p-value<0.01, *** indicates p-value<0.005.

Results in FIGS. 35-36 are shown as means plus standard error of themean (s.e.m.). p-value was calculated by Chi-squared test. * indicatesp-value<0.05, *** indicates p-value<0.001.

Short RNA Sequencing Data Statistical Analysis.

Statistical significance of downregulation of normalized miRNAs in Dicerand Drosha knockdown samples were calculated using the Wilcoxonsigned-rank test.

The differences in the fraction of 22-23 nt vs total short RNAs at thelocus between the wildtype, Dicer knockdown, and Drosha knockdown beforeand after cut was calculated by fitting a negative binomial model to thesRNA count data and performing a likelihood ratio test, keeping thefraction of 22-23 nt vs total short RNAs at the locus fixed acrossconditions under the null hypothesis and allowing it to vary betweenconditions under the alternative hypothesis.

LNA Sequences (for Experiments in FIGS. 34 and 36).

LNA 1: (SEQ ID NO: 57) TTATCCGCTCACAATTCCACAT LNA 2: (SEQ ID NO: 58)ATGTGGAATTGTGAGCGGATAA LNA 3 (Cntrl in FIG. 36): (SEQ ID NO: 59)ACTGATAGGGAGTGGTAAACT LNA 4: (SEQ ID NO: 60) AGAGAAAAGTGAAAGTCGAGTLNA 5 (control in FIG. 34): (SEQ ID NO: 61) CCCTAACCCTAACCCTAACCC LNA 6:(SEQ ID NO: 62) GGGTTAGGGTTAGGGTTAGGG

Examples

Inactivation of DICER and DROSHA Inhibits DDR and Allows Senescent Cellsto Re-Enter into Cell Cycle.

Oncogene-induced senescence (OIS) is a non-proliferative statecharacterized by a sustained DDR^(20,21) (caused by high level ofendogenous DNA damage) and senescence-associated heterochromatic foci(SAHF)²². Since the RNAi-machinery has been involved in heterochromatinformation²³, the authors investigated whether the inactivation ofcomponents of the RNAi machinery could have an impact on escape fromsenescence induced in human fibroblasts by transduction of H-RasV12(referred here as OIS cells). The authors therefore suppressed theexpression of DICER or DROSHA in OIS cells using a pool of smallinterfering RNAs (siRNA) and monitored cell-cycle progression intoS-phase with BrdU labeling. The authors observed that DICER or DROSHAknockdown, as well as ATM knockdown used as positive control for escapefrom senescence²⁰ (FIG. 51 a), result in an increased fraction ofBrdU-positive cells (FIG. 7B), re-expression of markers of chromosomalDNA replication (FIGS. 7C-7E) and entry into mitosis (FIGS. 7F-7G).These results could be reproduced over a range of siRNA concentrations(FIGS. 8A-8C) and with four individual siRNA oligonucleotides (FIGS. 8Dand 8E). Perhaps unexpectedly however, in DICER- or DROSHA-inactivatedcells the authors failed to detect any overt impairment inheterochromatin formation and SAHF components accumulation as detectedby DAPI staining and by immunostaining and immunoblotting for thetrimethylated form of the histone H3 (H3K9me3) (FIGS. 9A and 9B).

Since DDR plays a crucial role in the maintenance of the proliferativearrest in OIS cells^(20,21), the authors monitored whether DICER orDROSHA inactivation had an impact on DDR foci maintenance. The authorstherefore stained cells for markers of active DDR such as theautophosphorylated form of ATM (pATM), phosphorylated substrates of ATMand ATR (pS/TQ), 53BP1 and γH2AX. The authors observed that DICER orDROSHA inactivation significantly reduces the number of 53BP1, pATM andpS/TQ foci positive cells (FIGS. 1A, 1B, and 9A) even though 53BP1, ATMor H2AX protein levels are not reduced (FIG. 9C). The authors alsoobserved that the percentage of γH2AX-positive cells did not show asignificant variation, although the intensity of γH2AX foci wasgenerally reduced (FIGS. 1A and 1B). These effects could be observedover a range of siRNA concentrations (FIGS. 9D-9F). Importantly,inactivation of GW182/TNRC6A, the main component of the RNAi machineryinvolved in mRNA translational control, did not impact on DDR focidetection (FIGS. 10A and 10B), nor the simultaneous inactivation of allthree GW182-like proteins TNRC6A, B and C with two independent pools ofsiRNAs (FIGS. 11A and 11B). Therefore, DICER or DROSHA inactivationimpairs DDR signaling and overcomes the DDR-induced proliferative arrestof OIS cells.

DICER or DROSHA Inactivation Impairs Ionizing Radiation-Induced DDR FociFormation.

The authors next asked whether the involvement of DICER and DROSHA inDDR activation is specific for the senescence condition or whether DICERor DROSHA inactivation has also an impact on ionizing radiation(IR)-induced DDR activation in proliferating non-senescent cells.Therefore, the authors transiently inactivated DICER or DROSHA by a poolof siRNA in human normal fibroblasts (HNF—WI38; FIGS. 12A and 12B),exposed them to IR and a few hours later the authors stained them formarkers of activated DDR. The authors observed that, despite not reducedlevels of protein expression (FIG. 12C), formation of pATM, pS/TQ, MDC1,but not γH2AX, foci are impaired in DICER- or DROSHA-inactivated HNF(FIGS. 1C and 1D). These observations can be reproduced by using fourindividual siRNAs (FIGS. 12D-12G) and in a different HNF cell line (BJ,data not shown). Under these conditions (DICER- or DROSHA-knocked downHNF analyzed 7 hours post IR), the authors did not observe the dramaticimpairment of 53BP1 foci previously observed in OIS cells. However, ananalysis performed at earlier time points (10′ after IR) showed asignificant reduction of 53BP1 foci formation in DICER- orDROSHA-inactivated HNF (FIG. 13A), suggesting that DICER or DROSHAinactivation delays 53BP1 foci formation.

In order to exclude off target effects, the authors expressed anRNAi-resistant form of DICER in DICER-knocked down HeLa cells. Theauthors observed that re-expression of wild-type DICER, but of not amutant allele (DICER44ab) previously shown to abolish its RNAendonuclease activity²⁴, allows DDR foci formation to an extent similarto wild type cells, thus confirming DICER-dependency of the effectsobserved (Figure S 7b-d). Finally, the effects observed are independentof mRNA translational control, as GW182 knockdown has no significantimpact on DDR foci formation (FIGS. 14A-14C), consistent with theresults in OIS cells (FIGS. 10A and 10B). As an additional control, thesimultaneous inactivation of TNRC6A, B and C or DICER in Hela cellsexpressing a reporter mRNA encoding for Red Fluorescent Protein (RFP)carrying three binding sites for miR-126, used as a sensor formicroRNA-dependent translational repression, showed that both GW-likeproteins and DICER inactivation result in comparable RFP upregulation,due to the abolished miR-126-dependent RFP translational repression(FIGS. 15A, 15B, and 15D); nevertheless, only DICER inactivation affectsDDR foci stability (FIG. 15C).

To further confirm the involvement of DICER in DDR activation, theauthors used a colon cancer cell line (RKO) carrying a homozygoticgenetic deletion of exon 5 in DICER gene and therefore expressing ahypomorphic allele of DICER (DICER^(exon5)); this cell line is defectivein microRNAs maturation²⁵. In DICER^(exon5)-hypomorphic cells, the levelof expression of ATM, MDC1, 53BP1 or H2AX proteins is not reduced (FIG.16A). However, while in wild-type (WT) cells IR induces pATM, pS/TQ,MDC1 and γH2AX foci, in DICER^(exon5)-hypomorphic cells DDR fociformation is impaired (FIGS. 1E and 1F). In a time-course experiment,the authors observed that 53BP1 foci formation is delayed inDICER^(exon5)-hypomorphic cells (FIGS. 16B and 16C). Also in thissystem, the authors observed that γH2AX foci are only mildly affected(FIGS. 1E and 1F). The defects observed in DICER^(exon5)-hypomorphiccells could be reversed by the re-introduction of wild-type but not ofan endonuclease mutant allele of DICER (DICER44ab) (FIGS. 17A and 17B).Similar conclusions were reached in an additional cell line (HCT116)carrying the same DICER deletion (data not shown).

Next, the authors tested if the absence of DDR foci observed in DICER-or DROSHA-inactivated cells was due to a defect in actual DDR activationor DDR foci assembly. Therefore, the authors performed a set ofimmunoblot analyses both in DICER- or DROSHA-interfered HNF and inDICER^(exon5)-hypomorphic cell lines. The authors' analyses revealedthat IR-induced ATM autophosphorylation is impaired in DICER- orDROSHA-inactivated fibroblasts (FIGS. 18A and 18B) and in irradiated RKODICER^(exon5)-hypomorphic cells, compared to wild-type cells (FIG. 18C).This indicates that DICER and DROSHA control ATM activation and not justits accumulation in foci. Combined, these results reveal that DICER orDROSHA inactivation impairs DDR activation induced by exogenous sourcesof DNA damage in manner independent from canonical RNAi translationalrepressors (GW-like proteins).

DICER or DROSHA Inactivation Impairs G1/S and G2/M DNA DamageCheckpoints.

DNA damage elicits DDR signal transduction leading tocheckpoint-dependent cell-cycle arrest at two critical transition steps:the G1/S checkpoint and the G2/M checkpoint¹. The authors tested whetherimpaired DDR activation in DICER- or DROSHA-inactivated cells has animpact on G1/S and G2/M checkpoints. To test the G1/Scheckpoint-dependent arrest, cells were irradiated and pulse-labeledwith BrdU. The authors observed that DICER- and DROSHA-inactivated HNF(WI38; FIGS. 19A and 19B) have an impaired irradiation-inducedG1/S-phase arrest compared to control cells (FIG. 2a ) and that theextent of checkpoint deficiency is comparable to that of53BP1-interfered cells, used as a positive control²⁶ (FIGS. 2A and 19C).G1/S checkpoint impairment was confirmed with four individual siRNAoligonucleotides (FIGS. 19D and 19E) and in two additional HNF celllines (MRC-5 and BJ) in separate sets of experiments (FIGS. 19F and 19G,and data not shown). Consistent with the results in HNF,DICER^(exon5)-hypomorphic cells, from two distinct cell lines (RKO andHCT116; FIGS. 2B and 20A, respectively), also show an impaired G1/Stransition arrest. To confirm the dependency of the checkpoint on DICERin these cell lines, the authors re-expressed wild-type DICER-cDNA inDICER^(exon5)-hypomorphic cells. Indeed, DICER cDNA expression restoredthe G1/S checkpoint in both DICER^(exon5)-hypomorphic cell lines (FIGS.2B and 20A). Next, the authors asked if the RNA-endonuclease activity ofDICER is required for the DNA damage-induced checkpoint activation. Withthis aim, the authors complemented DICER^(exon5)-hypomorphic cells withtwo distinct DICER mutants carrying the amino acid substitution Asp44 toAla44 (DICER44ab) or Glu110 to Ala110 (DICER110ab) known to abolishDICER RNA endonuclease activity²⁴. While wild-type DICER expressionrescued the G1/S checkpoint defect of DICER^(exon5)-hypomorphic cells,both DICER mutants failed to do so (FIG. 2B), despite similar levels ofDICER expression (FIG. 20B). The authors conclude that the RNAprocessing activity of DICER is necessary to enforce the G1/Scheckpoint.

The authors also tested whether DICER is required to arrest cell-cycleprogression at the G2/M boundary following DNA-damage. Thus, the authorssuppressed DICER, or p53 as positive control, in HEK293 cells and theauthors tested G2/M checkpoint activation by monitoring the cell-cycleprogression profile over time through Fluorescence-Activated CellSorting (FACS). As expected, irradiated empty-vector (EV) transfectedcells progressively accumulate in the G2 phase of the cell cycle, as aconsequence of the checkpoint enforcement. Differently, DICER, as wellas p53 knocked-down cells, did not arrest upon DNA damage and passedthrough the G2/M transition (FIGS. 2C and 2D). This result was furtherconfirmed by monitoring the percentage of mitotic cells in control andDICER-inactivated cells following IR, based on histone H3phosphorylation (pH3) (FIGS. 20C and 20D)²⁷. Furthermore, the defect inG2/M checkpoint activation in DICER-knocked down cells could be rescuedby the expression of a siRNA-resistant DICER expressing construct (FIGS.20E-20G).

These results indicate that DICER-inactivated cells are deficient in theactivation of both G1/S and G2/M checkpoints and that DICER's RNAprocessing activity is necessary to enforce the checkpoint after DNAdamage.

DICER Inactivation in Zebrafish Impairs DDR Activation In Vivo.

To study if DICER is required for DDR activation upon irradiation in aliving organism, the authors tested the impact of DICER inactivation inDanio rerio (zebrafish) larvae, as a model system. Zebrafish embryoswere injected with morpholino oligonucleotides against Dicer1.Efficiency of Dicer1 inactivation was assessed by the ability of themorpholino oligonucleotide to block microRNA maturation and thereforeimpede the suppression of the co-injected reporter RFP-miR-126²⁸. Inaddition, the authors investigated the levels of six different maturemicroRNAs using QPCR to confirm inactivation of Dicer1. Larvaeoriginated from embryos injected with morpholino oligonucleotidesagainst Dicer1 displayed upregulated RFP expression and thedevelopmental defects previously reported for Dicer1-inactivatedlarvae²⁹ (FIGS. 21A and 21B) together with reduced levels of microRNAs(FIGS. 21C and 21D). Irradiated larvae were stained with antibodiesagainst pATM and γH2AX. Not irradiated larvae showed no or weak staining(FIGS. 3A-3C). Irradiation induced a strong pATM and γH2AX activation inall the cells throughout the sections of wild-type larvae head.Differently, irradiated Dicer1 morpholino-injected larvae showed adramatic impairment both in pATM and γH2AX signal (FIG. 3A). Theobserved impact on γH2AX suggests a stronger dependency of γH2AX onDicer1 in zebrafish in vivo, compared with cultured mammalian cells. Animmunoblot analysis of protein extracts from wild-type and Dicer1morpholino-injected larvae treated in parallel showed that theimpairment in pATM and γH2AX accumulation is present in the whole animal(FIG. 3B). The differential response to irradiation of cells withreduced Dicer1 activity due to morpholino injection versus Dicer1proficient cells was further confirmed in reciprocal celltransplantation experiments. Briefly, cells from embryos injected withDicer1 morpholino and mRNA encoding for GFP were transplanted atblastula stage into control uninjected embryos. Chimaeric larvae wereirradiated at 3 days post fertilization (dpf) and stained withantibodies against γH2AX (FIG. 3C). In the reciprocal experiment,control cells from embryos injected with mRNA encoding for GFP weretransplanted into Dicer1 morpholino injected embryos. Chimaeric larvaewere irradiated as above and stained with antibodies against γH2AX. Inboth cases, cells with reduced Dicer1 activity displayed reduced γH2AXsignals compared to their neighboring, Dicer1-proficient cells. (FIG.3C). The authors conclude that Dicer1 is required for DDR activation invivo in living zebrafish larvae.

In an In Vitro Cell System DDR Foci are Sensitive to RNase A and DICERand DROSHA RNA Products Allow DDR Foci Reformation.

The authors then sought an experimental system amenable for the study ofthe potential direct contribution of DICER and DROSHA RNA products inDDR activation. It has been previously shown that mammalian cells canwithstand a transient membrane permeabilization and RNase treatment.This approach allowed the study of the contribution of RNA toheterochromatin structure and protein association withchromatin^(30,31). The authors therefore utilised this technique toaddress the contribution of RNA in DDR activation. IR-exposed humancells (HeLa) were permeabilized by a mild detergent and treated with thebroad-specificity RNA nuclease RNase A. This treatment leads to thedegradation of both messenger RNAs and miRNAs including the mRNAs of DDRgenes (FIGS. 22A and 22B), without significantly affecting DDR proteinlevels (FIG. 22C). Untreated and RNase A-treated irradiated cells werestained for markers of DDR activation. The authors observed that RNAdegradation strongly impairs 53BP1, pATM, pS/TQ and MDC1 foci formation(FIGS. 4A and 4B). This result is consistent with the reportedsensitivity of 53BP1-GFP to ribonuclease treatment³¹. Similar to theauthors' observations in DICER- and DROSHA-inactivated cells, γH2AXaccumulation is only slightly affected by RNase A (FIGS. 4A and 4B).Noteworthy, unperturbed γH2AX signals indicate that RNase A treatmentdoes not dramatically alter chromatin structure or nuclear integrity.Intriguingly, 53BP1, MDC1 and γH2AX triple staining shows that RNase Areduces 53BP1 and MDC1 accumulation at individual γH2AX-foci, which areinstead maintained (FIG. 22D), thus suggesting that RNA molecules act tofavor MDC1 and 53BP1 focus formation once H2AX has been phosphorylated.

The authors also noticed that when RNase A is inactivated by an RNase Ainhibitor (RNaseOUT, a small protein inhibitor), DDR foci progressivelyreappear within minutes. In addition, the authors also observed thatfoci reformation can be prevented by the RNA polymerase II-specificinhibitor α-amanitin (FIGS. 23A and 23B). This suggests that that DDRfoci formation is dependent on RNA polymerase II RNA products.

Next, the authors tested if DDR foci that are lost after RNase Atreatment can reform following the addition of purified RNA to RNaseA-treated cells. Therefore, irradiated RNase A-treated cells werewashed, incubated with RNaseOUT and α-amanitin and incubated withHeLa-purified total RNA. Strikingly, the authors observed that theaddition of total RNA, but not tRNA used as control, robustly restoresfocal accumulation of all DDR factors tested (FIGS. 4D and 4E) within arelatively short time (15 minutes) at room temperature.

As IR may induce different kinds of DNA lesions, the authors expressedthe site-specific endonuclease PpoI^(32,33) which generates severalgenomic DSBs. Also in this system, the authors could demonstrate that53BP1, pATM and pS/TQ signals assemble in DDR foci that are sensitive toRNase A treatment and that their reformation can be induced by theaddition of RNA extracted from the same cells (FIGS. 24A-24C). Similarconclusions were reached when using an inducible form of the restrictionenzyme AsiSI³⁴ (data not shown).

Next, the authors attempted to characterize the RNA species involved inDDR-foci reformation by incubating RNase A-treated cells with differentRNA populations. To gauge the length of the RNA molecules involved inDDR focus reformation, the authors enriched total HeLa RNA for shortRNAs by chromatography (<200 nt; FIG. 25A) and the authors usedproportional volumes of total and short RNA to restore DDR foci in RNaseA treated cells. The authors observed that the short RNAs-enrichedfraction was sufficient to restore pATM, pS/TQ and 53BP1 foci indicatingthat this fraction contains the active RNA molecules (FIGS. 25B and 25C)To attain better RNA size separation, the authors resolved total RNA ona polyacrylamide gel and recovered RNAs of different lengths: longerthan 100 nt, between 100 nt and 35 nt and between 35 nt and 20 nt (FIGS.4C, 26A, and 26B). Using equal amounts of RNA from each fraction, theauthors observed that only RNAs in the 20-35 nt size range are active inrestoring DDR foci formation (FIG. 4C). Thus, two different separationapproaches indicate that RNA components required for DDR foci assemblyare short and in the size range of the RNA products generated by DICERand DROSHA.

Since the authors observed that inactivation of DICER and DROSHA affectsDDR foci formation in living cells and organisms, the authors reasonedthat its small RNA products could indeed be responsible for DDR focirestoration in this in vitro cell system. Thus, the authors investigatedif DICER RNA products directly contribute to DDR foci formation. To doso, the authors extracted total RNA from wild-type andDICER^(exon5)-hypomorphic cells and the authors used these two RNApreparations to restore DDR foci in RNase A-treated irradiated cells.Total RNA preparations from the two cell lines are expected to have thesame composition apart from the population of DICER RNA products²⁵.Strikingly, while RNA extracted from wild-type cells does restore pATM,pS/TQ or 53BP1 foci, RNA extracted from DICER^(exon5) hypomorphic cellsdoes not (FIGS. 4D and 4E). Importantly, RNA from DICER^(exon5)hypomorphic cells transfected with a vector expressing wild-type but notmutant DICER allows DDR foci reformation (FIGS. 27A and 27B). Theseresults can be quantitatively reproduced using RNA preparations from twoadditional cell lines (HCT116 and DLD1) carrying the same hypomorphicDICER mutation²⁵ (FIG. 28A) and by the use of RNA extracted from cellstransiently knocked-down for DICER (FIGS. 28B-28D). To test if also RNApurified from DROSHA-inactivated cells is unable to restore DDR foci,the authors knocked-down DROSHA, and GFP as control, by siRNA in HeLacells, purified RNA and used these RNA preparations to attempt torestore DDR foci. The authors' experiments revealed that total RNA fromsiGFP control cells restores DDR foci, while RNA purified fromDROSHA-inactivated cells does not (FIG. 28E).

Overall, these observations are consistent with a model in which smallRNA molecules generated by DICER and DROSHA are necessary to formIR-induced DDR foci. One conceivable mechanism is that small RNAproducts from DICER and DROSHA activity suppress the translation of ahypothetical DDR inhibitor. However, the observation that after RNase Atreatment (which degrades both mRNAs and microRNAs) (FIGS. 22A and 22B)and α-amanitin treatment (which inhibits transcription), gel-purified20-35 nt short RNA promote DDR foci reformation, strongly indicates thatDICER and DROSHA RNA products control DDR directly and independentlyfrom potential mRNA targets and translational modulation. Moreover, thetranslation inhibitor cyclohexamide has no impact on DDR foci formationin this system (data not shown). These conclusions are consistent withthe observation that inactivation of DICER or DROSHA, but not GW-likeproteins involved in translational inhibition, impacts on DDR activationin living cells.

DDR Focus Formation at a Defined Damaged Genomic Site Requires DamageSite-Specific RNAs.

Ionizing radiations induce the formation of DNA lesions that areheterogeneous in nature and random in their location. To reduce thisdiversity, the authors studied a single DSB at a unique, defined andtraceable genomic locus. The authors therefore took advantage of NIH2/4mouse cells carrying an integrated copy of the I-Sce I restriction siteflanked by an array of Lac-repressor (Lac) binding sites and Tetrepeats³⁵. In this system, the expression of the I-Sce I restrictionenzyme together with the fluorescent protein Cherry-Lac-repressor(Cherry-Lac) allows the visualization in the nucleus of thesite-specific DSB generated by the nuclease. Indeed, co-expression ofI-Sce I and Cherry-Lac-repressor in NIH2/4 cells induces a 53BP1 andγH2AX focus that overlaps with a focal Cherry-Lac signal (FIG. 5A). Theauthors observed that, also in this system, RNase-A treatment causes thedisappearance of the 53BP1 focus from I-Sce I-induced DSB (FIGS. 5A and5B) and α-amanitin prevents 53BP1 focus reformation at the same site(data not shown). Next, the authors tested if total RNA re-addition,following RNase A treatment, restores DDR focus at the I-Sce I-inducedDNA lesion. Therefore, RNase A-treated NIH2/4 cells were incubated withincreasing amounts of total RNA extracted from cells treated inparallel. When RNA was added to the RNase A-treated samples, NIH2/4cells re-acquired a bright 53BP1 focus co-localizing with theCherry-Lac-repressor in a manner dependent on the RNA amount used (FIGS.5A and 5B). Therefore, the very same DDR focus generated on a definedDSB can disassemble and reassemble in a manner dependent on RNA.Collectively, the results described so far demonstrate that DICER andDROSHA short RNA products control DDR foci formation. However, as such,they do not allow to discriminate whether RNAs are generated in cisusing the damaged genomic locus as a template or in trans from adistinct locus. To discriminate between these two possibilities, theauthors took advantage of the fact that the I-Sce I-induced DSB isgenerated within an exogenous sequence, which is not present in theparental cell line. The authors therefore transfected I-Sce I and theCherry-Lac-repressor in NIH2/4 cells and in the NIH3T3 parental cellline and the authors used RNA extracted from either cell lines toattempt to restore 53BP1 focus formation in RNase A-treated NIH2/4 cellsthat had experienced the I-Sce I-induced DSB: the two RNA preparationsare expected to differ only in the potential presence of RNA transcriptsgenerated from the exogenous integrated construct carrying the Lac andTet repeats and the I-Sce I site. Excitingly, the 53BP1 focus assemblyon the I-Sce I-induced DSB, as labeled by Cherry-Lac-repressor, wasefficiently recovered only by the addition of RNA purified from NIH2/4cells and not by RNA extracted from the NIH3T3 parental cell line (FIG.5C). This result indicates that DDR-focus formation requires an RNAcomponent, which originates from the damaged genomic locus.

The MRE11/RAD50/NBS1 (MRN) complex is a key DNA damage sensor and anecessary cofactor of the apical DDR regulator ATM¹. Also MRE11 focusformation upon I-Sce I induction is sensitive to RNase A-treatment (FIG.29A). To probe the molecular mechanisms of action of RNAs at sites ofDNA damage, the authors used mirin, a specific small molecule inhibitorof MRN³⁶ which, as expected, prevents ATM activation also followingI-Sce I induction (FIG. 29B). The authors therefore tested whether RNAsinvolved in DDR modulation engage MRN. The authors observed that in thepresence of mirin, NIH2/4 RNA is unable to induce 53BP1 or pATM focusreformation (FIGS. 5D and 5E). This result demonstrates that RNAs atsites of DNA damage modulate DDR in a MRN-dependent manner.

To detect potential short RNAs originating from the integrated locus,the authors isolated nuclear RNA from parental NIH 3T3 cells transfectedwith the I-Sce I (mock), NIH 2/4 cells transfected withCherry-Lac-repressor (uncut) and from NIH 2/4 cells transfected with theI-Sce I (cut) and further selected them for length (<200 nt)—thisprocedure enriches for RNAs active in DDR foci reformation 40 folds(data not shown). Libraries prepared from these samples were sequencedby Illumina GAII-X to obtain 15-32 bp cDNA reads (FIGS. 30A-30D). Theiranalyses revealed transcripts arising from the exogenous locus whichwere absent in the parental NIH 3T3 cells which had a lengthdistribution of mapped tags peaking around 22 nt, the length ofcanonical miRNAs (FIG. 29E). Thus, even an exogenous integrated locuslacking mammalian transcriptional regulatory elements is transcribed andgenerates short RNA transcripts.

In order to test whether the short RNAs identified at the damaged locusare biologically active in DDR activation, the authors chemicallysynthesized four potential pairs of short RNAs as identified at the cutgenomic locus by short RNA sequencing as described above and the authorsused them to attempt DDR focus reformation in RNase A-treated cellscarrying a DSB at the locus. By testing them over a large range ofconcentrations, the authors could demonstrate that the addition of theseRNAs, but not equal amounts of control ones, promote site-specific DDRactivation at the damaged site (FIG. 6A). These chemically-synthesizedshort RNAs are biologically active both when added to the cells togetherwith total RNA from parental cells (cells not carrying the integratedendogenous locus; FIG. 6A) or with yeast tRNA (FIG. 30F), thus in theabsence of any additional mammalian RNA.

In addition, to prove the biological activity of locally generated DICERRNA products, the authors cloned the locus, and an unrelated controlDNA, in a plasmid to allow its transcription in vitro by T7 polymeraseand the authors processed the resulting RNAs with recombinant DICERprotein in vitro. The resulting short RNAs (FIG. 30G) were purified andtested in RNase A-treated cells. The authors observed that also theselocus-specific DICER-generated RNAs, but not equal amounts of controlRNAs, allow DDR focus reformation both when mixed with RNA from parentalcells (FIG. 6B) and when mixed with yeast tRNA (FIG. 30H). Thus, invitro generated DICER RNA products promote DDR focus reformation at theDNA damaged site in a sequence-specific manner.

Overall, these results indicate that short RNAs with the sequence of thedamaged locus play a direct role in DDR activation at the damaged site.

In order to further investigate the nature of the RNAs generated at thelocus, the authors performed deeper sequencing experiments of nuclearRNAs <200 nt from wildtype NIH 2/4 samples before and after cut usingthe Illumina Hi seq Version3 (FIGS. 31A-31I). To study the biogenesis ofthese RNAs, the authors also sequenced <200 nt nuclear RNAs from NIH 2/4cells following Dicer or Drosha knockdown (WT uncut, Dicer KD uncut,Drosha KD uncut, WT cut, Dicer KD cut, Drosha KD cut) (FIG. 32A). Toease normalization, each RNA preparation was spiked with a short RNA“spike” before library preparation. Reads were mapped to miRBase 18 and,after spike normalization, were demonstrated to be significantly reducedafter Dicer or Drosha knockdown in uncut and cut samples when comparedwith wildtype uncut and cut samples respectively (FIG. 32B). Thisvalidates the functional efficacy of the knockdowns performed.

By analyzing the locus, the authors found that in wildtype samples thebulk of RNAs from the locus were in the 22-23 nt size range (45.2% in WTuncut and 67.6% in the wildtype cut, FIGS. 6C and 33A). Fitting anegative binomial model to the sequence count data and application ofthe likelihood ratio test showed that this increase in the fraction of22-23 nt vs total short RNAs in wildtype cut sample is statisticallysignificant respect to the uncut sample (p=0.020) (FIG. 6C). Further,the authors found that the fraction of 22-23 nt vs total short RNAs atthe locus decreases upon Dicer knockdown, both in the uncut (p=4.8e−7)and cut (p=0.029) conditions, suggesting that the 22-23 nt RNAs at thelocus are indeed Dicer dependent (FIG. 6C). The fraction of 22-23 nt vstotal short RNAs at the locus also decreases upon Drosha knockdown.

To further exclude that the majority of tags arising from the locus wereproducts of random degradation, the authors compared the fraction of22-23 nt vs total RNAs at the locus to the same fraction at non-miRNAgenomic loci—at such loci, any 22-23 nt RNAs are most likely products ofrandom degradation or Dicer/Drosha independent enzymatic processing. Theauthors found that the fraction of 22-23 nt vs total short RNAs issignificantly larger than the fraction found in non-miRNA genomic locibefore cut (p=0.049) and after cut (p=0.022, FIG. 33B).

Finally, the authors observed that the distribution of nucleotides atthe 5′ and the 3′ end of RNA sequences from the locus is significantlydifferent from both the genomic background nucleotide distribution(p=0.012 at the 5′ end and 0.008 at the 3′ end) as well as thebackground nucleotide distribution at the locus (p=0.014 at the 5′ endand 1.2e−6 at the 3′ end). Specifically, 82.9% sequences start with anA/U and 48.6% sequences end with a G (FIG. 6D).

By these analyses the authors therefore conclude that 22-23 nt RNAs arethe bulk of the RNA species detected at the locus, they depend on Dicerand, to an extent, on Drosha, and their proportion increases upon DNAdamage. Their unlikelihood to be random degradation products is furtherindicated by their differential abundance compared to the rest ofnon-miRNA loci and the observed 5′ and 3′ base bias.

Sequence-Specific Inhibitory Oligonucleotides (LNAs) Reduce DDRActivation at a Specific Genetic Locus.

The authors previously showed that DDRNAs identified by deep sequencingare biologically active and have a causative role in sequence-specificDDR focus reformation at the damaged site, following removal of allcellular RNAs by RNaseA treatment (FIG. 6a ; FIG. 300.

The authors next aimed to test whether DDRNA functions could beinactivated in living cells by sequence-specific Locked Nucleic Acids(LNA, modified DNA oligonucleotides avidly binding and inactivatingcomplementary RNA species) (Jepsen et al., Oligonucleotides, 2004;Machlin et al., Curr Gene Ther., 2012).

The authors thus got 4 LNA molecules synthesized (Exiqon) with theirsequence fully complementary to the individual DDRNAs they previouslyshowed to be biologically active and able to restore DDR signaling andfocus formation in RNaseA-treated cells (FIG. 6a ; FIG. 300. Given therepetitive DNA sequence structure of the locus, these are likely toanneal also to other DDRNAs generated at the locus (FIG. 34a ).

Cells carrying the integrated locus were co-transfected with Cherry-Lacand I-Sce I-restriction endonuclease-expressing vectors and with eitherno LNA (sample 1), control LNA carrying an unrelated sequence which isnot part of the locus (sample 2) or different sets of LNA (samples 3-7)(FIG. 34b ). 24 hours post transfection, cells were fixed and stainedfor DDR markers. The authors analyzed the samples at the confocalmicroscope and scored as positive those cells that showed a DDR signalat the locus. As shown in FIG. 34B, the portion of cells showing aspecific γH2AX focus co-localizing with the Cherry-Lac signal was notsignificantly affected by the transfection of any LNA. This isconsistent with the authors' data showing that any impairment of thebiogenesis of DDRNAs or removal of RNAs by RNaseA treatment makes nosignificant impact on γH2AX (FIG. 1; FIGS. 4A, 4B, 4D, and 4E; FIG. 5A).Differently, 53BP1 accumulation at the locus, a marker of activated DDR,is significantly reduced upon transfection of LNA with the sequencematching the DDRNAs (samples 3-6), compared to control LNA (sample 2) orto the same LNA inactivated (sample 7; FIG. 34C). LNA were inactivatedby annealing them to each other in vitro before transfection, thusbecoming unable to bind and interfere with the action of othercomplementary nucleic acids. The decrease in 53BP1 accumulation wasobserved, to different extents, for all LNA sets tested (samples 3-6).In summary, these results demonstrate that sequence-specific LNAs canspecifically inactivate the known biological functions of DDRNAs.

Sequence-Specific Inhibitory Oligonucleotides (i.e. LNAs) with aTelomeric Sequence Reduce DDR Activation at Dysfunctional Telomeres.

The authors previously showed that short RNAs with the sequence of adamaged locus (named DDRNAs) are necessary for DDR activation andmaintenance specifically at that locus, upon ionizing radiations orendonuclease cleavage (FIGS. 4,5). However, nothing is known about therole of DDRNAs at telomeres. Telomeres are the end of linearchromosomes, and they are protected by a protein complex named shelterin(de Lange, Genes Dev. 2005). Removal of this protection causes telomereuncapping, so telomeres are recognized as DNA DSBs. This may lead to DDRactivation, cellular senescence, chromosomal fusions and genomeinstability (Sfeir and de Lange, Science 2012).

In order to investigate the role of DDRNA at dysfunctional telomeres,the authors used CRE-ER TRF2^(flox/flox) mouse embryonic fibroblasts(MEFs) (Lazzerini Denchi and de Lange, Nature 2007). Cells were grown inpresence of 4-hydroxytamoxifen to induce cre recombinase localizationinto the nucleus, thus generating a TRF2-knockout (TRF2^(−/−)) cellline. TRF2 is one of the shelterin component and its removal induces astrong DDR activation at telomeres (Lazzerini Denchi and de Lange,Nature 2007). To test the role of DDRNA at the telomeres, we treatedMEFs TRF2^(−/−) with RNase A or BSA as a control. Consistent with theauthors' previous results, they observed that γH2AX foci resist, while53BP1 foci are sensitive to RNase A treatment (FIG. 35a-b ). Thissuggests that, like all other DSB lesions, also at uncapped telomeres,DDRNAs with telomeric sequence are generated and they are necessary forDDR cascade activation.

Cells can accumulate damaged telomeres during ageing, due to telomericshortening (Harley, Nature 1990; Herbig, Mol Cell 2004; d'Adda diFagagna, Nature 2003) or to endogenous or exogenous DNA damage occurredat telomeres. DNA damage accumulate and DDR signaling persists attelomeres as they are not repairable (Fumagalli, Nat Cell Biol 2012). Inboth cases this persistent DDR activation at telomeres leads to cellularsenescence. If DDRNAs are necessary for DDR at damaged telomeres,inhibiting their action could suppress DDR activation and potentiallyprevent or revert the senescence phenotype. To test this hypothesis, theauthors used a human cell line, T19 fibrosarcoma cells, that express aninducible dominant negative (DN) allele of flag-tagged TRF2 (vanSteensel, Cell 1998). The expression of this allele is induced culturingcells in the absence of doxycycline. After 7-8 days of DN TRF2expression, telomeres are dysfunctional and cells show a strong DDRactivation at telomeres (data not shown and van Steensel, Cell 1998).Induced cells, in which Flag-DN TRF2 was expressed, are positive forFlag immunostaining (Flag+). At day 13 from induction, the authorstransfected T19 cells with LNA molecules with sequences complementary toeither strands of telomeric DNA repeats (LNA 5 and 6) or an unrelatedsequence as control (LNA 3, Cntrl). The authors observed that, withtime, in Flag+ cells, both LNAs transfected individually with telomericsequence decrease the percentage of 53BP1-positive cells, to a differentextent, while control LNA had no effect (FIG. 36a ). Importantly, inuninduced undamaged cells (Flag−), LNA molecules are not inducing anyDNA damage, excluding that they can be genotoxic per se. Furthermore,the authors monitored the passage through S phase of cell cycle, lookingat BrdU incorporation in induced T19 cells, three days after LNAtransfection. The authors observed that Flag+cells, transfected withtelomeric LNAs, proliferate significantly more than control cells (FIG.36b ), suggesting that LNA, by inactivating DDR at telomeres, canpromote cell cycle reentry of cells otherwise activating a DNA-damagecheckpoint and reducing BrdU incorporation. In contrast there is nosignificant difference in Flag− cells (FIG. 36c ).

Here the authors show that different sources of DNA damage, includingoncogenic stress, ionizing irradiation and PpoI or I-Sce I endonucleasesengage the DDR in a manner dependent on DICER and DROSHA RNA products.These DDR-regulating RNAs (DDRNAs) control DDR-foci formation andmaintenance, checkpoint enforcement and cellular senescence. This occursboth in cultured human and mouse cells and in different cell types inliving zebrafish embryos.

Oncogene activation can trigger DDR and DDR-induced cellular senescenceacts as tumor suppressive mechanism^(2,37). DICER inactivation enhancestumor development in a K-Ras-induced mouse model of lung cancer^(38,39)and inactivation of various components of DICER and DROSHA complexesstimulate cell transformation and tumorigenesis³⁸. More recently,mutations of DICER and TARBP2, a DICER cofactor affecting its stability,have been described in human carcinomas^(40,41). However, individualmicroRNAs have been reported both to promote and to reduce cellproliferation by regulating stability and translation of mRNAs encodingproteins with different roles in cell proliferation¹⁸: it is thereforepresently unclear how RNAi apparatus inactivation favors tumorigenesis.In the light of the authors' novel findings pointing to a role of DDRNAsin DDR control, a known tumor suppressive mechanism³⁷, it is tempting tosuggest that, in addition to their well-characterized functions in themodulation of gene expression, DICER and DROSHA RNA products may curbcancerous cell proliferation by sustaining DDR activation and thisgenerates the selective pressure for the inactivation of factorsinvolved in their biogenesis. The authors also report that in an invitro cellular system, DDR foci are lost in irradiated cells followingRNase A treatment and that site-specific DDRNAs, even if generated bychemical synthesis or upon in vitro cleavage by recombinant DICER, arerequired to restore them. This suggests that DDRNAs are locallygenerated and favor the assembly of DDR factors in the shape ofdetectable DDR foci at the DNA damaged site. Indeed RNA sequencingconfirmed the presence of short RNAs arising from the integratedexogenous locus which are induced upon cut. Comparison with short RNAsgenerated at other non miRNA genomic loci indicates that they aredistinct from products of RNA degradation and their nucleotide bias at5′ end and 3′ end indicates that these RNAs are processed atpreferential RNA precursors sites.

Although at present how DDRNAs act to control DDR activation has notbeen elucidated in full, the observation that they act in a mannerdependent on the MRN complex place them upstream of the canonical DDRsignaling cascade.

Although novel and unanticipated, the authors' results are consistentwith the emerging evidence supporting a role for RNA molecules in DDR.Indeed, an epistasis map generated in fission yeast has recently shownthat DDR components display genetic interactions with the RNAimachinery⁵⁶ and components of the large DROSHA complex have beenidentified in a ATM-dependent phosphoproteome screen⁵⁷. In Drosophila,repeated DNA integrity is dependent on RNAi pathway⁵⁸. In Saccharomycescerevisiae and in Oxytricha Trifallax RNA orchestrates recombination andRNA can function as a template for DNA repair events in S. cerevisiae^(59,60,61) It is also intriguing to observe that like several DDRfactors, that are inactivated early in apoptosis in order to prevent DDRactivation⁶², also DICER is specifically cleaved by caspases duringapoptosis⁶³. Recently, ATM has been shown to directly modulate thebiogenesis of DICER and DROSHA RNA products by phosphorylating KSRP⁶⁴.

Finally, it is worth noticing that the here-described novel functions ofcomponents of the RNAi machinery in the modulation of the response toDNA damage are consistent with its well-established andevolutionary-conserved role of preserving genome integrity from viralinvaders, transposons and retroelements⁶⁵.

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1. A method of detecting damage to DNA in a sequence-specific genomiclocus in a cell comprising: detecting the presence of small RNAs(DDRNAs), said small RNAs being generated by processing by DICER and/orDROSHA of a RNA transcript synthesized upon transcription of the damagedgenomic locus in said cell; and comparing the result to a control cellwith undamaged DNA genomic locus.
 2. A method of identifying the genomiclocation of damage to DNA in a sequence-specific genomic locus in a cellcomprising: isolating and/or purifying small RNAs (DDRNAs) from asample, said small RNAs being generated by processing by DICER and/orDROSHA of a RNA transcript synthesized upon transcription of the damagedgenomic locus in said cell; and sequencing said isolated and/or purifiedsmall RNAs (DDRNAs).
 3. (canceled)
 4. The method of claim 1, whereindetecting the presence of the DDRNAs comprises measuring an amount ofthe DDRNAs; and comparing the result to a proper control, wherein thecomparison to the proper control allows diagnosing and/or prognosing acondition associated with and/or induced by generation of DNA damage inat least one sequence specific genomic locus.
 5. The method according toclaim 4, wherein the condition associated with and/or induced by thegeneration of DNA damage in at least one sequence specific genomic locusis selected from the group consisting of: cancer, aging and viralinfection.
 6. The method according to claim 5 wherein aging isassociated with critically short and/or damaged and/or dysfunctionaltelomeres.
 7. The method of claim 4, further comprising: afteradministration of a therapy for a condition associated with and/orinduced by the generation of DNA damage in at least one sequencespecific genomic locus, measuring an amount of the DDRNAs; and comparingthe result to a proper control, wherein the comparison to the propercontrol allows for monitoring efficacy of the therapy.
 8. The methodaccording to claim 7 wherein the condition associated with and/orinduced by the generation of DNA damage in at least one sequencespecific genomic locus is selected from the group consisting of: cancer,aging and viral infection.
 9. The method according to claim 8 whereinaging is associated with damaged telomeres.
 10. A method of screeningfor an agent able to inhibit small RNAs (DDRNAs), said small RNAs beinggenerated by processing by DICER and/or DROSHA of a RNA transcriptsynthesized upon transcription of a damaged genomic locus in a cellcomprising measuring an amount of said small RNAs upon exposure of thecell to said agent, and comparing to a proper control.
 11. The method ofclaim 1, wherein the cell is a mammalian cell.
 12. The method of claim11, wherein the mammalian cell is a human cell.
 13. The method of claim12, wherein the cell is selected from the group consisting of apre-cancerous, cell, a cancer cell, a senescent cell and a viral cell.14. The method of claim 13, wherein the senescent cell has criticallyshort and/or damaged and/or dysfunctional telomeres.
 15. The method ofclaim 2, wherein the cell is a human cell.
 16. The method of claim 15,wherein the cell is selected from the group consisting of apre-cancerous, cell, a cancer cell, a senescent cell and a viral cell.17. The method of claim 16, wherein the senescent cell has criticallyshort and/or damaged and/or dysfunctional telomeres.
 18. The method ofclaim 10, wherein the cell is a human cell.
 19. The method of claim 18,wherein the cell is selected from the group consisting of apre-cancerous, cell, a cancer cell, a senescent cell and a viral cell.20. The method of claim 19, wherein the senescent cell has criticallyshort and/or damaged and/or dysfunctional telomeres.